alpha 1-Fetoprotein mRNA of rat yolk sac and hepatoma.

Rat alpha 1-fetoprotein mRNA was isolated and purified to apparent homogeneity by means of immunoadsorption and oligo (dT) cellulose affinity chromatography. Purified AFP mRNA migrated as a 21S peak in 2.5% SDS-polyacrylamide gels. The translation product of this mRNA in micrococcal nuclease treated reticulocyte lysate was identified as AFP by specific immunoprecipitation, SDS-gel electrophoresis and tryptic digestion analysis. DNA complimentary to AFP mRNA was synthesized with avian meyloblastosis virus RNA-dependent DNA polymerase. This AFP cDNA was used as a probe to quantitate AFP mRNA in the developing rat liver and to compare the complexity and diversity of AFP mRNA derived from the normal rat liver and Morris hepatoma 7777. We found that the amount of functional AFP mRNA is decreasing during liver development. There is very little, if any, AFP mRNA in the adult rat liver. A high degree of homology between the AFP mRNA sequences of yolk sac and hepatoma was also found.     

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.     

A specific alpha-fetoprotein gene binding protein in alpha-fetoprotein producing rat hepatomas

A specific alpha-fetoprotein (AFP) gene binding nuclear protein (Mol. Wt. 149,000) was determined in Morris hepatoma 7777 cells by the protein blotting technique. This protein is not present in normal adult rat liver and non-AFP producing Morris hepatoma 5123tc. Neoplasia induced in rats fed the hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzine enhanced AFP gene activity and re-expressed specific AFP gene binding nuclear protein. The precise role of this protein in AFP gene regulation remains to be determined.     

Cloning of rat alpha-fetoprotein 3'-terminal complementary deoxyribonucleic acid sequences and preparation of radioactively labeled hybridization probes from cloned deoxyribonucleic acid inserts

Double-stranded complementary deoxyribonucleic acid (cDNA) was synthesized from rat yolk sac alpha-fetoprotein (AFP) mRNA, inserted into the PstI site of plasmid pBR322 by an oligo(deoxyguanylic acid).oligo(deoxycytidylic acid) joining technique, and cloned in Escherichia coli chi 1776. A plasmid containing an inserted AFP double-stranded cDNA with a contiguous poly(adenylic acid) [poly(A)] segment was identified and subsequently employed in a new method for preparing AFP-specific hybridization probe. Following an initial digestion of the AFP plasmid with HindIII to create an open, recessed 3' end, lambda exonuclease III was employed to remove the DNA strand opposite the coding strand of the cDNA insert. Oligo(thymidylic acid) was then annealed to the poly(A) segment and employed as primer for E. coli DNA polymerase I to synthesize a 32P-labeled cDNA copy of the AFP coding strand. The single-stranded cDNA product was easily isolated by sedimentation through isokinetic alkaline sucrose gradients. Hybridization with this AFP-specific cDNA probe showed that the yolk sac contained a 6-fold greater concentration of AFP mRNA than that of the fetal liver. AFP mRNA was also found in the normal adult liver, but at a much lower level than in the fetal liver. The concentrations of AFP mRNA in Morris hepatomas 7777 and 8994, however, were significantly elevated to a 2- to 3-fold higher concentration that in the fetal liver.     

Effects of hexamethylene bisacetamide onα-fetoprotein,albumin, and transferrin production by two rat hepatoma cell lines

Summary α-Fetoprotein (AFP), albumin, and transferrin production by two rat hepatoma cell lines, McA-RH 7777 (7777) and McA-RH 8994 (8994), was determined after treatment with hexamethylene bisacetamide (HMBA, 2 to 6 mM). Radioimmunoassays were used to determine the levels of both secreted and intracellular AFP, albumin, and transferrin. Line 7777 normally produces large quantities of AFP and small quantities of albumin, thus resembling the less differentiated fetal liver with respect to the synthesis of these two proteins. Line 8994 normally produces small quantities of AFP and relatively larger amounts of albumin, thus resembling hepatic functions characteristic of a more differentiated state. After treatment with HMBA for a period of 28 to 96 h a threefold increase in AFP secretion by 7777 and a dose related increase in AFP, albumin, and transferrin secretion by 8994 were observed. In contrast, the secretion of albumin and transferrin in 7777 was inhibited by 60 and 40%, respectively, following treatment with HMBA. The intracellular concentrations of AFP in 7777 and AFP, albumin, and transferrin in 8994 were increased by treatment with HMBA indicating that HMBA is able to stimulate the synthesis of these proteins. The intracellular concentration of AFP, albumin, and transferrin in 7777, when expressed as a percentage of the extracellular concentration of these proteins, did not change significantly during HMBA treatment, indicating that the observed decrease in secreted albumin and transferrin by 7777 is due to decreased synthesis. Similarly, in Line 8994, when the intracellular concentration of the three proteins was expressed as percentage of the extracellular concentration, the only significant change observed was an increase in AFP after 72 h of HMBA (5 mM) treatment. The observed changes in the synthesis of AFP, albumin, and transferrin in both 7777 and 8994 after HMBA treatment were reversible, as judged by the return to control values upon removal of HMBA from the culture medium. Thus, HMBA stimulates synthesis of the oncofetal protein AFP, a result that appears to be independent of the stage of differentiation of the cell. However, its effect on the synthesis of albumin and transferrin are opposite in the two cell lines, suggesting that the regulation of the synthesis of these two proteins is controlled by factors or conditions that are dependent upon the stage of differentiation of the hepatoma cell lines.     

Isolation of rat liver albumin messenger RNA.

Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.     

Segregation of specific classes of messenger RNA into free and membrane-bound polysomes

Analysis of mRNA populations from rat liver rough microsomes and free polysomes by homologous and heterologous cDNA . mRNA hybridization shows that the two mRNA populations are distinct, demonstrating that specific mRNA classes are efficiently segregated for translation in association with endoplasmic reticulum membranes. We estimate that approximately 90% of the mRNA in membrane-bound polysomes contains a diverse set of messengers with a minimum of 500--2000 different species although approximately 5--8 messengers may constitute 25--30% of the mRNA mass. The complexity of the mRNA population of free polysomes appears to be comparable to that estimated for total liver poly(A) + mRNA by other investigators, and is likely to be substantially greater than that of the bulk of bound mRNA. In addition, mRNA in free polysomes lacks the high abundance class characteristic of mRNA-bound polysomes. The substantial complexity of the bound mRNA population suggests that the segregation of polysomes in rough microsomes is not limited to a small class specialized in manufacturing secretory proteins, but extends to polysomes engaged in the synthesis of proteins for intracellular distribution. The segregation of specific messengers into the free and membrane-bound classes was abolished when polysome disassembly was induced by administration of ethionine. Thus, messenger RNA molecules themselves lacked the capacity for segregation, although they contain information for segregation which is expressed during translation. These findings are consistent with the presence of signal sequences in nascent polypeptides which determine the attachment of ribosomes to endoplasmic reticulum membranes.     

Identification of albumin-synthesizing polysomes from mouse liver and a mouse hepatoma cell line.

Albumin-synthesizing polysomes from mouse liver and mouse hepatoma cells in in tissue culture have been localized on sucrose gradients with ¹²⁵I-labeled antimouse serum albumin used as a marker. Competition studies show that the ¹²⁵I-labeled antibody binds specifically to albumin-synthesizing polysomes from both tissues. The ¹²⁵I-labeled polysomes from liver and hepatoma cells have identical sedimentation properties on sucrose gradients, which indicates that the polysomes range in size from 9–14 ribosomes. This is comparable in size to polysomes from rat liver and Morris hepatoma. One significant difference between these albumin-synthesizing polysomes is that those extracted from hepatoma cells bind 70% less antibody than equivalent amounts of polysomes from liver cells. Since the level of albumin synthesis in the hepatoma cells is comparable to the level of albumin synthesis $\text{in vivo}$, this difference in antibody-binding capacity is not likely to be due to differences in polysomal content, but appears to be a characteristic difference between hepatoma and normal mouse liver cells.     

A complete complementary DNA for the oncodevelopmental calcium-binding protein, oncomodulin

RNA from a rat liver tumor (Morris hepatoma 5123tc) was used to construct cDNAs together comprising the complete coding sequence of rat oncomodulin mRNA. Information obtained from these cDNAs as well as from primer extension analysis gave a deduced length for the complete oncomodulin mRNA of approximately 680 nucleotides (excluding the poly(A) tail) including a 5'-untranslated region of 97 +/- 2 nucleotides, a 324-nucleotide-coding sequence and a 259-nucleotide 3'-noncoding region. Comparison of the oncomodulin cDNA sequence with those coding for other members of the calcium-binding protein family shows little homology with the exception of a recently reported parvalbumin cDNA where the oncomodulin and parvalbumin nucleotide sequences are 59% identical in the protein-coding region. RNA blot analysis of poly(A+) RNA from normal adult rat liver gave no evidence of oncomodulin expression in this tissue. A single RNA species was detected, however, in RNA extracts from the hepatoma and from rat and human placentas. A probe prepared from one of the rat oncomodulin cDNAs hybridized with a single DNA species in restriction digests of hepatoma and normal DNA from rat and sequences in DNA of humans and other mammals. A 38-nucleotide sequence spanning the 5'-untranslated region and the first seven codons of the oncomodulin cDNA, was far less homologous than was the same region of a parvalbumin cDNA, to a chicken calmodulin cDNA sequence coding for the first calcium-binding domain. The oncomodulin gene appears to have diverged more from that of calmodulin than has the parvalbumin gene.     

The expressions of oncogenes and liver-specific genes in Morris hepatomas

The expression of three liver-specific genes and four oncogenes was studied in the Morris hepatomas 8994, 7288c, 7777, 5123tc, and 7800. Total RNA isolated from these tumors was probed with cDNA's for alpha-fetoprotein (AFP), albumin, tyrosine aminotransferase (TAT), and the oncogenes Ha-ras, Ki-ras, myc and src. When compared to mRNA's levels expressed in normal adult liver, we found AFP levels elevated in AFP-producing tumors, albumin and TAT mRNA levels depressed in all tumors, except TAT is elevated in 5123tc and the oncogenes with the exception of src elevated in all tumors. These results argue against a coordinated expression of these genes as a result of transformation, but suggest that oncogene expression is related to tumorigenesis or proliferation.     

Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

Mutations were introduced in 7 kilobases of 5'-flanking rat alpha 1-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.     

Purification of messenger RNA coding for glycine methyltransferase from rat liver

Rat liver messenger RNA coding for glycine methyltransferase was associated preferentially with free polysomes. The mRNA was purified about 1000-fold over the total poly(A)-containing RNA by specific immunoadsorption of polysomes to protein A-Sepharose followed by oligo(dT)-cellulose column chromatography. Sodium dodecyl sulfate-gel electrophoresis of the in vitro translation products in a rabbit reticulocyte lysate system revealed only one major band which migrated to the position of the purified glycine methyltransferase subunit. The result shows that the mRNA isolated is nearly homogeneous and suggests that no precursor form of the enzyme existed. The mRNA sedimented at the position slightly smaller than 18 S rRNA in a sucrose density-gradient centrifugation and was shown to contain about 1,300 nucleotides by the Northern blot hybridization analysis with a cDNA probe.     

Complex population of nonpolyadenylated messenger RNA in mouse brain

The complexity of nonadenylated mRNA [poly(A)-mRNA] has been determined by hybridization with single-copy DNA (scDNA) and cDNA. Our results show that poly(A)- and poly(A)+ mRNA are essentially nonoverlapping (nonhomologous) sequence populations of similar complexity. The sum of the complexities of poly(A)+ mRNA and poly(A)- mRNA is equal to that of total polysomal RNA or total mRNA, or the equivalent of approximately 1.7 x 10(5) different sequences 1.5 kb in length. Poly(A)- mRNA, isolated from polysomal RNA by benzoylated cellulose chromatography, hybridized with 3.6% of the scDNA, corresponding to a complexity of 7.8 x 10(4) different 1.5 kb sequences. The equivalent of only one adenosine tract of approximately 20 nucleotides per 100 poly(A)- mRNA molecules 1.5 kb in size was observed by hybridization with poly(U). cDNA was transcribed from poly(A)- mRNA using random oligonucleotides as primers. Only 1-2% of the single-copy fraction of this cDNA was hybridized using poly(A)+ mRNA as a driver. These results show that poly(A)- mRNA shares few sequences with poly(A)+ mRNA and thus constitutes a separate, complex class of messenger RNA. These measurements preclude the presence of a complex class of bimorphic mRNAs [that is, species present in both poly(A)+ and poly(A)- forms] in brain polysomes.     

The degree of methylation of alpha-fetoprotein in rat hepatoma cell lines and their clones producing and not producing this protein

The extent of methylation of MspI-HpaII sites of alpha-fetoprotein (AFP) gene in DNAs from normal rat livers, rat hepatoma cell lines and their clones was compared by Southern blot hybridization. The expression of this gene was controlled by measuring the level of AFP specific RNA and by immunoperoxidase staining with antibodies to AFP. It was found that the AFP gene contained several CCGG sites in the 5'-region methylated in the nonproductive cell lines and normal adult liver and undermethylated in productive cell lines. The same phenomena was observed for productive and nonproductive hepatoma McA-RH7777 clones. These findings suggest that specific changes in the methylation pattern are associated with changes in AFP gene expression.     

Phosphatidylserine biosynthesis in mitochondria from the Morris 7777 hepatoma.

Mitochondria from the 7777 hepatoma incorporate substantial amounts of l-[U-(14)C]serine into phospholipid by a Ca(2+)-dependent base-exchange reaction. This reaction is virtually absent in normal liver mitochondria. The finding cannot be attributed to microsomal contamination of the sucrose gradient-purified 7777 hepatoma mitochondria. The reaction is also absent in the rapid-growth controls, fetal rat liver and regenerating rat liver. [(14)C]Serine incorporation into 7777 hepatoma mitochondrial phospholipid by base-exchange requires Ca(2+) and is inhibited by EDTA. Ca(2+) cannot be replaced by Mg(2+), Mn(2+), or Co(2+). The reaction is inhibited by a sulfhydryl reagent and by detergents and is abolished by heating to 70 degrees C for 10 min. Product analysis indicates that phosphatidylserine and its decarboxylation product, phosphatidylethanolamine, are formed by 7777 hepatoma mitochondria, while phosphatidylserine is the sole product with microsomes. The conversion of phosphatidylserine into phosphatidylethanolamine in 7777 hepatoma mitochondria is inhibited by KCN. This study provides further evidence of abnormal mitochondrial biogenesis in the 7777 hepatoma. Our earlier study indicated a greatly increased mitochondrial activity of CTP:phosphatidate cytidylyltransferase in the 7777 hepatoma (Hostetler, Zenner, and Morris. 1978. J. Lipid Res. 19: 553-560). The presence in mitochondria of these two enzymes, which are primarily microsomal in normal liver, does not appear to be due to rapid growth alone, since their intracellular distribution was not altered in fetal or regenerating rat liver.-Hostetler, K. Y., B. D. Zenner, and H. P. Morris. Phosphatidylserine biosynthesis in mitochondria from the Morris 7777 hepatoma.