Α-fetoprotein mRNA Research Articles

In situ hybridization analysis of α-fetoprotein mRNA expression in rat liver during regeneration after partial hepatectomy

In situ hybridization analysis of α-fetoprotein mRNA expression in rat liver during regeneration after partial hepatectomy

Hepatic Level of Rat Albumin Messenger RNA is Influenced by Factors other than Dietary Protein

Dot hybridization of messenger RNA (mRNA) and complementary DNA (cDNA) has been used to measure the relative levels of albumin and α-fetoprotein mRNA in liver of rats fed for 5 d a fat-free (carbohydrate-rich) diet, a high fat diet or a basal diet, all three of which were isonitrogenous. The level of albumin mRNA in rats fed the fat-free (carbohydrate-rich) diet was 30 to 45% of the level in animals fed the basal 4% fat diet. The concentration of another mRNA, that for α-fetoprotein, remained unchanged. It has been established by others that albumin mRNA levels and albumin synthesis are diminished in response to low levels of dietary protein. We show that albumin mRNA levels are lower than those observed in animals fed the basal 4% fat diet, even when dietary protein is adequate (30% wt/wt), if the nonprotein calories are derived solely from carbohydrate.

The ontogeny of expression of murine metallothionein: Comparison with the α-fetoprotein gene

The ontogeny of expression of mouse metallothionein was studied by RNA dot and Northern blot hybridization using a cloned cDNA probe. In some instances the synthesis of metallothionein was analyzed by cell-free translation of RNA as well as pulse-labeling of proteins in short-term organ cultures followed by polyacrylamide gel electrophoresis. Interesting parallels between metallothionein and α-fetoprotein gene expression during development were noted. Like α-fetoprotein mRNA (Dziadek and Andrews, 1983), metallothionein mRNA was found to be abundant in developing liver as well as in visceral yolk sac endoderm. In addition, metallothionein mRNA was abundant in parietal yolk sac. During liver development metallothionein and α-fetoprotein mRNAs were abundant by Day 12 of gestation, increasing to maximal levels on Day 16 and decreasing during late fetal and neonatal life to basal levels in adult. Metallothionein mRNA increased in maternal liver and was also abundant in certain hepatomas. Synthesis of metallothionein and levels of metallothionein mRNA in visceral yolk sac increased from Day 9 of gestation to maximal levels on Days 11–12 and then decreased abruptly after Day 15. RNA from differentiated teratocarcinoma cells with primitive, parietal or visceral endoderm characteristics each contained high levels of metallothionein mRNA, whereas, levels of this mRNA varied widely among embryonal carcinoma stem cell lines. α-Fetoprotein mRNA was not detected in embryonal carcinoma cells but was expressed in visceral endoderm-like differentiated cells. These results indicate that parietal and visceral endoderm cells actively express the metallothionein gene and further suggest that expression may be initiated at the earlier stage of primitive endoderm.

Selective detection of rat and mouse specific albumin and α-fetoprotein mRNA molecules under highly stringent hybridization conditions

The molecular analysis of some important interactions observed between the parental genomes in interspecific cell hybrids requires the availability of highly specific hybridization assays to selectively quantitate mRNA sequences coding for the same protein but transcribed from the two different genomes. Specific hybridization techniques which should permit the selective detection of rat and mouse albumin and α-fetoprotein (AFP) mRNA molecules in a mixture of the two types of mRNAs are presented here. The high degree of homology existing between the AFP mRNA sequences coding for mouse and rat AFP, and, presumably, albumin, results in extensive cross-hybridization with the cDNA probes under standard hybridization conditions. No size differences could be detected between the two types of mRNA molecules from the two species. A T m difference of 7 °C between the intra- and interspecific mRNA:rat cDNA hybrids allowed the establishment of highly stringent solution hybridization conditions necessary to measure separately the contents of rat albumin and AFP mRNAs. Mouse albumin and AFP cDNA clones were then isolated from mouse liver and yolk sac cDNA libraries, and used to show the usefulness of highly stringent washing conditions to discriminate between rat and mouse albumin and AFP mRNA molecules in conventional “Northern blotting” techniques. In combination with the solution hybridization assay, these filter hybridization techniques can be used to specifically quantitate the content of rat and mouse albumin and AFP mRNA molecules in interspecific cell hybrids.

Distribution of mRNAs of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of α-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5–6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2–3-fold). There were no α-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.

Sequence content of α-fetoprotein, albumin and fibrinogen polypeptide mRNAs in different organs, developing tissues and in liver during carcinogenesis in rats

To investigate the variable gene activities of α-fetoprotein, albumin and fibrinogen polypeptides as markers of ‘liver specific proteins’ in different developing organs or tissues, we have used specific complementary DNA probes to detect and to quantitate α-fetoprotein, albumin and fibrinogen polypeptide mRNA, respectively, in RNA fractions, prepared from various tissues of rats at different stages of fetal and postnatal development and from hepatomas induced by diethylnitrosamine. The results indicate that there is no consistent relationship between sequence content of α-fetoprotein, albumin and fibrinogen polypeptide mRNA in different developing tissues. Intestines which are like the liver also of endodermal origin do not contain α-fetoprotein, albumin and fibrinogen polypeptide mRNAs, while kidneys which are mesodermal in origin were found to be α-fetoprotein, albumin and fibrinogen polypeptide mRNA producers in neonatal life. In yolk sac, only α-fetoprotein and fibrinogen polypeptide mRNA could be detected. In the liver, the increased level of albumin and fibrinogen polypeptide mRNA during fetal and neonatal development is accompanied with a diminished amount of α-fetoprotein mRNA. The neosynthesis of α-fetoprotein mRNA in the liver during carcinogenesis occurred without a decreased content of albumin and fibrinogen polypeptide mRNAs. These findings suggest that complex mechanisms of gene regulation are involved in variable gene activities of α-fetoprotein, albumin and fibrinogen polypeptides in cells of different organs or tissues developed from a single cell.

Molecular cloning of DNA complementary to a mouse α-fetoprotein mRNA sequence

DNA complementary to mouse yolk sac messenger RNA has been inserted at the PstI site of the plasmid pBR322 by annealing of the oligo(dG)-tailed plasmid DNA with the oligo(dC)-tailed mouse DNA. Transformation of Escherichia coli strain RRI with this annealed DNA yielded clones bearing recombinant plasmids. The clones were screened for DNA complementary to mouse a-fetoprotein (AFP) messenger RNA sequences by hybridization with a cDNA probe transcribed from an AFP mRNA of over 90% purity. Out of nine plasmids that were isolated and analyzed by restriction mapping, all had homologous insert DNA of various lengths. The plasmid with the longest insert, pAF6, contained 1.65 kb of added DNA, which is about 70% of the AFP mRNA. This clone was positively identified by a hybridization-translation procedure to contain a cDNA sequence for AFP. A restriction map of this clone and the orientation of the message are presented.

Dexamethasone inhibits α-fetoprotein gene expression in developing mouse liver

The effect of dexamethasone on α-fetoprotein gene expression was studied in newborn mice. The treatment of 3 day old mice with 1 μg/gm body weight of dexamethasone twice a day reduced the α-fetoprotein level in serum to about 2.5% of the normal control level after 4 days of administration. The level of α-fetoprotein mRNA sequences in the cytoplasm decreased to about 1% of the control over this same period. Furthermore, only an extremely low concentration of α-fetoprotein mRNA sequences in nuclear RNA could be detected even at a high C rot. These results suggest that dexamethasone inhibits transcription of the α-fetoprotein gene in newborn mice.