Project: Research project

Project Details


The overall goals of the research are to understand the factors
regulating the content of the polyamines spermidine and spermine
in mammalian cells and to use this knowledge to manipulate cellular
polyamine levels and evaluate the role of the polyamines in normal
and neoplastic growth. The proposed investigations will focus
predominantly on S-adenosylmethionine decarboxylase (AdoMetDC)
although some studies on the regulation of ornithine decarboxylase
(ODC) by polyamines will also be carried out. Previous work
supported by this grant has led to the production of monospecific
antibodies to these enzymes and of cDNA clones for AdoMetDC.
Radioimmunoassays for these proteins and means of labelling their
active sites have also been developed. These tools and reagents
will be used for the proposed studies. The specific aims of the research proposal are: (1) To obtain the
complete cDNA sequence corresponding to rat AdoMetDC mRNA and to
derive amino acid sequence of AdoMetDC and its precursor. (2) To
investigate the biosynthesis and processing of the AdoMetDC
precursor using antisera to AdoMetDC to precipitate the relevant
proteins from a reticulocyte lysate protein synthesis system
supplemented with the mRNA. (3) To investigate the regulation of
ODC and AdoMetDC protein levels and synthesis rates by polyamines.
(4) To continue the study of the effects of polyamines on the
translation of the mRNA for AdoMetDC and ODC which we have found
to be much more sensitive to inhibition by polyamines than other
mammalian mRNAs. (5) To obtain CHO cell mutants lacking AdoMetDC
and use these to study the regulation of the synthesis of the
enzyme by transfection of constructs containing the AdoMetDC gene.
(6) To obtain genomic clones for AdoMetDC and use them to study the
regulation of enzyme synthesis. (7) To isolate and characterize
cDNA clones for AdoMetDC from human and trypanosome sources. Our previous work has shown that AdoMetDC from rat prostate
contains a covalently bound pyruvate prosthetic group and that the
enzyme is synthesized as a precursor of Mr 37,000 which is
converted to the enzyme sub-unit of Mr 32,000 in a reaction which
presumably generates this prosthetic group. The proposed work will
delineate the biochemical mechanism underlying the synthesis of
this enzyme and will provide new information on the regulation of
its biosynthesis.
Effective start/end date12/31/894/30/00