Masters Theses

Date of Award

12-1983

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Allen R. Chilina

Committee Members

Koontz, Richter, Riggsby

Abstract

The purpose of this study was to isolate, purify and compare polysomal poly(A)-containing mRNA from diethylnitrosamine induced primary hepatoms to polysomal poly(A)-containing mRNA from partial hepatectomized rat livers. Gross characteristics such as polysomal profiles, polysomal RNA, whole cell RNA, and purified polyadenylated mRNA profiles were similar. From 3H Poly(U) profiles of the polyadenylated mRNA, size distributions and number-average-nucleotide lenghts were determined. For DEN induced hepatomas and regenerating rat livers the mean sedimentation value and number-average-nucleotide lengths of the purified polyadenylated mRNA populations were similar and calculated to be approxmately 16.6S or 1700 nucleotides. The regenerating rat liver contained approximately 1.2 times more polyadenylated mRNA per gram of rat liver compared to the DEN induced hepatomas. The mean length of the 3' poly(A) tracts was 20% greater for the regenerating rat liver polyadenylated mRNA compared to the neoplastic tissue mRNA population.

Both RNA populations could direct the programming of polypeptides in the rabbit reticulocyte lysate in vitro translation system. Approximately 70% of the polysomal translational activity was found to be associated with the polyadenylated mRNA fraction, while 30% was associated with the non polyadenylated mRNA fraction. Many similarities existed between the neoplastic and regenerating rat liver polyadenylated mRNA translated products separated on polyacrylamide gels. However, quantitative and qualitative differences were observed. In particular the translated hepatoma mRNA had increased concentrations of proteins with molecular weights of 63000, 28000 and 26000 daltons. Also at least 2 unique proteins were observed in translated hepatoma mRNA compared to translated regenerating mRNA with molecular weights of 41000 and 30000 daltons. Full-length complementary DNA probes were synthesized to the polyadenylated mRNA populations, and the kinetics of homologous hybridizations were analyzed. The frequency and complexity of DEN induced hepatoma and regenerating rat liver polysomal polyadenylated mRNA populations were determined. Both polyadenylated mRNA populations were distributed into three frequency classes. The regenerating rat liver polyadenylated mRNA populations contained more highly repetitive RNA sequences compared to the neoplastic mRNA, 38% versus 26%. However, the neoplastic tissue had 44% moderately repetitive polyadenylated mRNA sequences compared to 34% for the regenerating polyadenylated mRNA sequences. The DEN induced hepatoma polysomal polyadenylated mRNA had a total sequence complexity of 1.64 x 1010 daltons commpared to a total sequence complexity for regenerating rat liver polyadenylated mRNA of 8.5 x 109 daltons. This information was used to calculated the total number of polyadenylated mRNA species present in both tissues; 31000 and 15000 polyadenylated mRNAs for neoplastic and regenerating tissues respectively.

One way of checking the calculated total complexity values was by utilizing homologous saturation hybridization experiments of single copy DNA to polyadenylated mRNA popultions from DEN induced hepatomas and regenerating rat liver. From this saturation hybridization experiment its was determined that there was enough DNA complexity to code for approximately 38000 and 20000 distinct 1800 nucleotide sequences for DEN induced hepatoma and regenerating rat liver polyadenylated mRNA species. It, therefore, appears that the DEN induced hepatomas have approximately twice the complexity and number of polyadenylated mRNA species compared to polyadenylated mRNA from regenerating rat livers. From the experiments performed, identifiable differences exist in DEN induced hepatoma and regenerating rat liver polyadenylated mRNA populations.

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