The cellular oncogene c-myc is expressed in many cell types. c- myc expression is highest in proliferating cells and it is turned off in differentiating cells. A human c-myc mRNA half-life of 30-60 minutes has been reported for several cell lines. In these studies, however, poly(a+) RNA prepared from whole cells was used as a source of c-myc RNA and not total cellular RNA. We have investigated the turnover of c-myc mRNA using total cellular RNA from the human promyelocytic leukemia cell line, HL-60. We find two classes of c-myc RNA, a relatively labile, polyadenylated population (which binds to oligo(dT)-cellulose) and a more stable c-myc RNA population lacking long polyadenylate (which does not bind to oligo(dT)-cellulose). Further structural analysis of these two RNA populations demonstrates that both have similar structures except that the labile, polyadenylated c- myc mRNA is approximately 200nt larger than the stable c-myc RNA which does not bind to oligo(dT)-cellulose. Taken together data suggest that poly(A) is a structural feature of c-myc mRNA that regulates its steady-state level. We will further study these two c-myc mRNA populations for their role in c-myc gene regulation. Subcellular localization of these RNAs will be accomplished by fractionating nuclei and cytoplasm. Questions we will ask are the following: 1) Are (A-) c-myc RNAs found in the nucleus or cytoplasm? 2) If found in the cytoplasm, are the (A) c-myc RNA species translated? We will also investigate the transcriptional and post-transcriptional regulation of both the (A-) and (A+) c-myc RNAs during mitogenic stimulation of fibroblasts (up regulation) and differentiation of HL-60 cells (down regulation). To investigate the nucleotide sequences involved in the regulation of c-myc expression fusion genes have been created. These fusion genes have structure, 5' c-myc/3' beta-globlin or 5' beta-globin/3' c-myc These fusion genes as well as various deletions and site- specific mutants will be introduced into cultured cells. c-myc RNA half-life studies and analysis of adenylation status will be performed. The role of poly(A) in c-myc RNA will be directly tested with fusion genes which have a transcribed poly(dT) tract inserted in the 3' untranslated region of the terminal c-myc exon. Resultant c-myc RNAs will be assayed for polyadenylation and RNA half-life. Transcriptional activation will be studied using a c-myc promoter/CAT gene fusion genes as well as 5' myc/3' beta- globin constructs.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA043661-03
Application #
3185959
Study Section
Molecular Biology Study Section (MBY)
Project Start
1987-07-15
Project End
1991-03-31
Budget Start
1989-07-01
Budget End
1991-03-31
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Roswell Park Cancer Institute Corp
Department
Type
DUNS #
City
Buffalo
State
NY
Country
United States
Zip Code
14263
Kinniburgh, A J; Firulli, A B; Kolluri, R (1994) DNA triplexes and regulation of the c-myc gene. Gene 149:93-100
Kolluri, R; Torrey, T A; Kinniburgh, A J (1992) A CT promoter element binding protein: definition of a double-strand and a novel single-strand DNA binding motif. Nucleic Acids Res 20:111-6
Baer, M R; Augustinos, P; Kinniburgh, A J (1992) Defective c-myc and c-myb RNA turnover in acute myeloid leukemia cells. Blood 79:1319-26
Kolluri, R; Kinniburgh, A J (1991) Full length cDNA sequence encoding a nuclease-sensitive element DNA binding protein. Nucleic Acids Res 19:4771
Kolluri, R V; Manuelidis, L; Cremer, T et al. (1990) Detection of monosomy 7 in interphase cells of patients with myeloid disorders. Am J Hematol 33:117-22
Kinniburgh, A J (1989) A cis-acting transcription element of the c-myc gene can assume an H-DNA conformation. Nucleic Acids Res 17:7771-8
Davis, T L; Firulli, A B; Kinniburgh, A J (1989) Ribonucleoprotein and protein factors bind to an H-DNA-forming c-myc DNA element: possible regulators of the c-myc gene. Proc Natl Acad Sci U S A 86:9682-6
Williams, S C; Grant, S G; Reue, K et al. (1989) cis-acting determinants of basal and lipid-regulated apolipoprotein A-IV expression in mice. J Biol Chem 264:19009-16
Cutry, A F; Kinniburgh, A J; Krabak, M J et al. (1989) Induction of c-fos and c-myc proto-oncogene expression by epidermal growth factor and transforming growth factor alpha is calcium-independent. J Biol Chem 264:19700-5
Swartwout, S G; Kinniburgh, A J (1989) c-myc RNA degradation in growing and differentiating cells: possible alternate pathways. Mol Cell Biol 9:288-95

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