Expression of mRNAs for alpha-fetoprotein (AFP) and albumin and incorporation of AFP and docosahexaenoic acid in baboon fetuses.

alpha-Fetoprotein and albumin, two members of a multigene family, reversibly bind fatty acids with high affinity. The origin of alpha-fetoprotein (AFP) and albumin present in fetal tissues other than the liver and yolk sac is a subject of controversy. In this work, we have searched for the presence of the albumin and AFP mRNA molecules in different fetal organs of the baboon (Papio cinocephalus), using a highly sensitive gel-blot hybridization assay with human albumin and AFP cDNA probes.

Binding of a liver-specific factor to the human albumin gene promoter and enhancer.

A segment of 1,022 base pairs (bp) of the 5'-flanking region of the human albumin gene, fused to a reporter gene, directs hepatoma-specific transcription. Three functionally distinct regions have been defined by deletion analysis: (i) a negative element located between bp -673 and -486, (ii) an enhancer essential for efficient albumin transcription located between bp -486 and -221, and (iii) a promoter spanning a region highly conserved throughout evolution. Protein-binding studies have demonstrated that a liver trans-acting factor which interacts with the enhancer region is the well-characterized transcription factor LF-B1, which binds to promoters of several liver-specific genes.

The organization of repetitive sequences in the albumin and alpha-fetoprotein gene loci in the rat.

The distribution of middle repetitive sequences in the genic and extragenic regions of the rat albumin and alpha-fetoprotein genes was analyzed. Their presence was determined by probing Southern blots of restriction fragments of albumin and alpha-fetoprotein genomic subclones with 32P-labeled total rat DNA. Repetitive sequences were detected in both genes.

Albumin and alpha-fetoprotein gene expression in various nonhepatic rat tissues.

Albumin and alpha-fetoprotein (AFP), two major serum proteins, are synthesized predominantly in the liver and yolk sac of mammals. In the present paper we report on the developmental expression of the corresponding genes in nonhepatic rat tissues. Significant quantities of mature albumin and AFP mRNAs were revealed in kidney, pancreas, heart, and lung of fetal and/or newborn rats using dot blot and Northern blot assays.

Differences in methylation patterns of the alpha-fetoprotein and albumin genes in hepatic and non hepatic developing rat tissues.

By use of different restriction enzymes sensitive to internal cytosine methylation (HpaII, AvaI, HhaI) we have analysed the methylation patterns of albumin and AFP genes in tissues and cell lines with high (liver, yolk sac, hepatoma cell lines), low (fetal and neonatal kidney) or undetectable (spleen, JF1 fibroblasts) expression of either gene. We show that expression of the AFP gene is associated to the demethylation of a whole region or domain extending from -4 to +3 Kb. Moreover, demethylation of a site located at the upstream limit of this domain appears to be correlated with the commitment of the cell type to synthesize AFP.

The rat alpha-fetoprotein and albumin genes. Transcriptional control and comparison of the sequence organization and promoter region.

Functional and structural approaches were used to characterize the transcription units of the rat alpha-feto-protein (AFP) and albumin genes. A cell-free nuclear transcription assay and several genomic clones were used to show that: 1) the rate of transcription of these genes is closely related to the levels of corresponding mRNAs in the yolk sac and during rat liver development, indicating that the expression of the albumin and AFP genes is mainly regulated at the transcriptional level in the rat, and 2) the in vivo 5' end boundaries of the rat AFP and albumin transcription domains were mapped near the respective first exons. Due to the presence of repeated sequences, the 3' end boundary of both genes could not be accurately defined in the same manner.

Differential stability of the higher order structure of chromatin associated with genes having different transcriptional activity.

We have investigated the stability of the higher order structure of chromatin associated to genes which display a different transcriptional activity in adult rat live. Nuclei were digested with micrococcal nuclease and chromatin was fractionated by sedimentation in sucrose gradients. Specific DNA sequences were revealed by dot-blotting.

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression.

Specific sets of DNase I-hypersensitive sites are associated with the potential and overt expression of the rat albumin and alpha-fetoprotein genes.

We have examined the chromatin structure of the 5'-flanking region of the albumin and alpha-fetoprotein (Afp) genes in different developing rat tissues and cloned cell lines that display various functional states of these genes. Nuclease-hypersensitive sites were probed with DNase I, using an indirect end-labeling technique. In albumin-producing rat cells two major DNase I-hypersensitive sites were found near the promoter region and one additional site was located approximately 3 kilobases (kb) upstream.

Phenobarbital-mediated modulation of gene expression in rat liver. Analysis of cDNA clones.

Phenobarbital evokes a pleiotypic response in the liver characterized by cell hypertrophy and mono-oxygenase induction. These phenomena arise through complex modulation mechanisms changing the pattern of protein synthesis, distinct from those triggered by other well known inducers, like steroid hormones or polycyclic hydrocarbons. To investigate the mechanisms involved in regulating the expression of the phenobarbital-inducible tissue-specific genes, we constructed two libraries of recombinant bacterial plasmids pBR322 in Escherichia coli.

Cellular analysis by in situ hybridization and immunoperoxidase of alpha-fetoprotein and albumin gene expression in rat liver during the perinatal period.

To analyze at the cellular level the decrease in alpha-fetoprotein (AFP) gene expression during the early postnatal growth, we searched for AFP gene transcripts by in situ hybridization using a specific cDNA probe, and for the corresponding protein by immunocytochemistry, on rat liver sections at various times of the perinatal period. The relative number of mRNA sequences was evaluated by Northern blot analysis. Albumin (ALB) gene expression was studied simultaneously with the same techniques.

Activation of alpha-fetoprotein synthesis in rat hepatoma cells with reduced sensitivity to dexamethasone.

The Faza 967 'differentiated', dexamethasone (DEX)-sensitive cell line of Reuber rat hepatoma cells does not synthesize detectable amounts of alpha-fetoprotein (AFP), whereas it does produce albumin. AFP production was activated in 'differentiated' variants of Faza 967 cells with reduced glucocorticoid sensitivity upon culture for several months in the presence of high concentrations of dexamethasone. The stability of AFP production differed among the variants, while albumin synthesis did not change, thus indicating that the regulation of these two genes is not co-ordinated.

Human inter-alpha-trypsin-inhibitor: characterization and partial nucleotide sequencing of a light chain-encoding cDNA.

A synthetic Inter-alpha-Trypsin-Inhibitor (ITI) -specific oligonucleotide probe was used to isolate a clone from a human liver cDNA library. The amino-acid sequence deduced from partial nucleotide sequencing of the corresponding cDNA insert perfectly matched a known ITI sequence, apart from an as yet unreported C-terminal dipeptide. Hybridization on Northern blots evidenced that this cDNA insert originated from an ITI light chain-encoding mRNA whose size was estimated to be 1 300 bases.

A lethal deletion on mouse chromosome 7 affects regulation of liver-cell-specific functions: posttranscriptional control of serum protein and transcriptional control of aldolase B synthesis.

Steady-state levels of mRNAs were determined for the serum proteins albumin, alpha-fetoprotein (AFP), and transferrin, as well as for aldolase B in livers of newborn mice homozygous for a radiation-induced lethal deletion (c14CoS) in chromosome 7. Deficiencies in synthesis and secretion of the serum proteins as well as in activities of certain liver-specific enzymes characterize these homozygotes. The results of RNA dot and gel-blot hybridizations with the respective cloned cDNA probes showed a decrease to one-fourth of aldolase B mRNA levels in homozygous mutant livers compared to normal littermates, in contrast to normal levels of mRNA sequences for the three serum proteins in the mutants.

Structural basis for restriction-site polymorphism at the albumin locus in inbred strains of rats.

Two types of variant EcoRI restriction enzyme patterns of albumin-gene DNA fragments are found in different inbred strains of rats and reflect allelic polymorphism. The structural basis of the two allelic forms has been analyzed by mapping the EcoRI fragments using cloned albumin cDNA probes corresponding to the 5' or 3' end of the rat albumin mRNA and different genomic subclones. Additional restriction fragment length polymorphism has been detected using the restriction endonucleases HindIII and MspI.

All hepatocytes are involved in the expression of the albumin gene in the normal adult rat: a demonstration by in situ hybridization and immunoperoxidase techniques.

Immunoperoxidase techniques have yielded conflicting results concerning the percentage of hepatocytes engaged in albumin production in normal adult rats. In addition, the question of whether functional differences in the synthesis of plasma proteins exist within the hepatic lobule remains to be determined. To clarify these questions, we have searched for gene albumin transcripts by in situ hybridization, and for the corresponding protein by immunoperoxidase, on adjacent liver sections.

Organization of the albumin and alpha-fetoprotein genes in fetal and adult rat tissues, and rat hepatomas.

We compared the organization of the albumin and alpha-fetoprotein (AFP) genes in chromosomal DNA from different fetal and adult rat tissues as well as from two rat hepatomas. These two genes are expressed at widely different levels in the tissues and hepatomas analysed. Southern blots of DNAs digested with the restriction endonucleases EcoRI, HindIII or MspI were hybridized to albumin and AFP complementary DNA (cDNA) and genomic probes.

DNA restriction polymorphism of alpha-fetoprotein gene after digestion by MspI endonuclease.

Hybridization of alpha-fetoprotein clones cDNA with human DNAs digested by seven restriction endonucleases reveals one polymorphism. This polymorphism, detected after restriction by MspI, is at low frequency (f = 0.02) and is validated by family analysis.

Differential DNase I sensitivity of the albumin and alpha-fetoprotein genes in chromatin from rat tissues and cell lines.

We have examined the DNase I sensitivity of the albumin and alpha-fetoprotein (AFP) genes in different rat tissues (adult liver and kidney) and cloned cell lines (hepatoma 7777-C8, JF1 fibroblasts), which show drastic differences in the level of expression of these two genes. This was done by studying the disappearance of defined restriction endonuclease fragments of these genes as a function of limited DNase I digestion. The sensitivity of these genes was compared to that of a gene not expressed in the hepatic cells and to that of a ubiquitously expressed gene.

Analysis of fibrinogen genes in patients with congenital afibrinogenemia.

Several cDNA clones coding for A alpha, B beta and gamma chains of fibrinogen have been isolated from a human liver cDNA library. They were selected by differential hybridization with probes raised against fractionated liver mRNA (positive probes) and muscle and albumin mRNA (negative probes), then firmly identified by positive hybridization selection. Three of these clones, encoding A alpha, B beta and gamma fibrinogen chain sequences, were further characterized by restriction mapping and used as probes to characterize fibrinogen mRNAs from adult and fetal liver and fibrinogen genes in normal individuals and two afibrinogenemic patients.

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.

Selective detection of rat and mouse specific albumin and alpha-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 alpha-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.

Structural variants of the alpha-fetoprotein gene in different inbred strains of rat.

Two structural variants of the rat alpha-fetoprotein (AFP) gene have been detected in different inbred strains of rats by EcoRI or HindIII restriction enzyme cleavage of cellular DNA, agarose gel electrophoresis and Southern blot hybridization using 32P-labeled cloned rat AFP cDNA probes. The type I AFP gene variant is characteristic of the Sprague-Dawley strain, and type II is found in Buffalo rats. These variants appear to represent two different allelic forms of the rat AFP gene since they are inherited in a normal Mendelian fashion when Sprague-Dawley and Buffalo rats are crossed.

Polymorphisms of human albumin gene after DNA restriction by HaeIII endonuclease.

Hybridization of albumin clones cDNA with human DNAs digested by several restriction endonucleases reveals two HaeIII polymorphisms. The first polymorphism, H1, is of low frequency (f1 = 0.05); the second, which is validated by family analysis, occurs frequently (f2 = 0.

Nucleotide sequence of a cDNA clone for human aldolase B.

Two specific clones for human aldolase B were isolated from a human liver cDNA library using a rat aldolase B cDNA probe. The clones were identified by positive hybridization-selection and one of them was sequenced. The 127 C-terminal residues of the human protein were deduced from this nucleotide sequence analysis.