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Changes in expression of albumin and alpha-fetoprotein genes during rat liver development and neoplasia
Abstract:
Albumin mRNA was isolated and purified from rat liver polysomes by a combination of immunoprecipitation of specific polysomes, poly(U)-Sepharose 4B chromatography, and fractionation of the resulting poly(A)-containing RNA on a sucrose gradient. alpha-Fetoprotein (AFP) mRNA was isolated from Morris hepatoma 7777 by a similar procedure. The purity of the mRNA preparations was determined by analytical gel electrophoresis under denaturing conditions, analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides synthesized in a wheat germ cell-free system, and the kinetics of hybridization to cDNA transcribed from albumin mRNA and AFP mRNA. The albumin mRNA possessed a chain length of approximately 2265 nucleotides and the AFP mRNA possesed a length of approximately 2235 nucleotides when examined under stringent denaturing conditions on agarose gels containing 10 mM methylmercury hydroxide. Analysis of poly(A) content by a hybridization assay with [3H]poly(U) revealed the presence in albumin mRNA of a poly(A) region containing approximately 100 adenosine residues. The AFP mRNA preparation was found to contain an average poly(A) tract of approximately 190 bases. Thus, albumin mRNA appears to contain approximately 330 untranslated nucleotides, and AFP mRNA appears to contain a similar number (approximately 285) of noncoding, nonpoly(A) bases. The purified albumin and AFP mRNA's were used as templates for synthesis of full-length cDNA hybridization probes. Both of the probes selectively hybridized to their templates with kinetics expected for single RNA species the sizes of albumin and AFP mRNA. ROt analysis was used to quantitate albumin and AFP mRNA sequences during normal liver postnatal development and liver oncogenesis. The number of polysomal AFP mRNA molecules per liver was found to drastically decrease during the first weeks of postnatal life, concomitant with a decline in the AFP synthetic capacity of the livers and in the serum concentrations of AFP. During this period, the concentration of albumin mRNA molecules per cell in the liver remained at high, approximately constant levels. In Morris hepatoma 7777, the concentration of AFP-specifying sequences was at least 10(3)-fold higher than that found in normal adult liver, whereas the content of albumin nRNA was four- to five-fold lower. These changes in concentration of albumin and AFP mRNA sequences closely correlated with a parallel variation in the specific protein synthetic capacity of the tissues.
Insights
Researchers purified albumin and alpha-fetoprotein (AFP) mRNA from rat liver and hepatoma, analyzing their lengths and poly(A) tails. AFP mRNA levels decreased postnatally, while albumin mRNA remained stable, with significant AFP mRNA elevation in hepatoma.
Area of Science:
- Molecular Biology
- Biochemistry
- Oncology
Background:
- Albumin and alpha-fetoprotein (AFP) are key liver proteins with distinct developmental and oncogenic expression patterns.
- Understanding the regulation of their respective messenger RNAs (mRNAs) is crucial for deciphering liver function and disease.
Purpose of the Study:
- To isolate and purify albumin mRNA from rat liver and AFP mRNA from Morris hepatoma 7777.
- To characterize the size, poly(A) tail length, and sequence complexity of these mRNAs.
- To quantify changes in albumin and AFP mRNA levels during normal liver development and hepatocarcinogenesis.
Main Methods:
- Isolation of polysomal mRNA via immunoprecipitation, poly(U)-Sepharose chromatography, and sucrose gradient fractionation.
- Purity assessment using gel electrophoresis, cell-free protein synthesis, and cDNA hybridization kinetics.
- Quantification of mRNA sequences using ROt analysis and hybridization to synthesized cDNA probes.
Main Results:
- Purified albumin mRNA (approx. 2265 nucleotides) and AFP mRNA (approx. 2235 nucleotides) were obtained, with distinct poly(A) tail lengths (albumin: ~100, AFP: ~190 bases).
- During normal liver development, AFP mRNA levels decreased significantly postnatally, while albumin mRNA levels remained constant.
- In Morris hepatoma 7777, AFP mRNA concentration was >1000-fold higher, and albumin mRNA was 4-5 fold lower than in normal liver.
Conclusions:
- The study successfully isolated and characterized albumin and AFP mRNAs, providing insights into their molecular properties.
- Differential regulation of albumin and AFP mRNA abundance is evident during liver development and oncogenesis.
- Changes in mRNA levels strongly correlate with the protein synthetic capacity of the liver tissues.