The Srp40 protein plays a dose-sensitive role in preribosome assembly or transport and depends on its carboxy-terminal domain for proper localization to the yeast nucleoskeleton.

The yeast SRP40 gene product (Srp40p) is a highly serine-rich protein organized in three distinct domains. The roles of these domains in localizing Srp40p were determined. By indirect immunofluorescence microscopy, Srp40p localizes to punctate, sometimes fibrillar, subnuclear structures that might include the nucleolus. Its amino-terminal and medial domains are similar. They each start with a short basic stretch containing a nuclear localization signal, followed by a long acidic stretch with 76% serines; such acidic stretches are thought to mediate binding to ribosomal proteins in the nucleolus. Either domain is sufficient to determine nuclear localization of Srp40p. The Srp40p carboxy-terminal domain shows significant homology to the cognate domain of Nopp140, a mammalian nucleolar phosphoprotein of 140 kD. The carboxy-terminal domain alone, or fused to a reporter protein, displays a punctate localization outside the nucleus. Srp40p and Nopp140 share a highly homologous 39-residue motif within their similar carboxy-terminal domains. Inside or outside the nucleus, this motif is important to prevent Srp40p diffusion or degradation. These observations suggest that the punctate immunoreactive structure is nucleoskeletal and might result from Srp40p self-assembly. SRP40 genetically interacts with four mutants affected in stable RNA synthesis and one mutant blocked in protein translocation to the endoplasmic reticulum. Growth defects, but no translocation or rRNA transcription/maturation phenotypes, were observed upon SRP40 inactivation or strong overexpression. Together, these data point to a dispensable, dosage-sensitive, role of Srp40p in preribosome assembly or transport.

Chicken ovalbumin upstream promoter-transcription factor, hepatocyte nuclear factor 3, and CCAAT/enhancer binding protein control the far-upstream enhancer of the rat alpha-fetoprotein gene.

We have further characterized the most distal of the three alpha-fetoprotein (AFP) enhancers required for expression of the AFP gene in fetal hepatocytes and yolk sac endodermal cells. Almost total rat AFP enhancer 3 (E3) activity is driven by a 160-bp fragment at -6 kb containing three target regions for nuclear proteins that cooperate to stimulate transcription from the AFP and the thymidine kinase promoters in HepG2 hepatoma cells. Region 1, recently shown to be crucial for correct function of the enhancer in liver of transgenic mice, is recognized by two sets of transcription factors that bind to partly overlapping sites, 1a and 1b, in a noncooperative and nonexclusive manner. Site 1a contains a motif, AGGTCA, which is recognized by chicken ovalbumin upstream promoter transcription factors (COUP-TFs), but not by hepatocyte nuclear factor 4. Hepatocyte nuclear factor 3 (HNF3) and CCAAT/enhancer binding protein (C/EBP), which bind to regions 2 and 3, respectively, are likely responsible for the liver-specific E3 action. They play a key role by acting in synergy. The participation of nuclear receptors such as COUP-TFs, with C/EBP and HNF3, in the tight control of the distal AFP enhancer is a new, and perhaps key, step toward understanding the regulation and function of this enhancer, which may remain active throughout development.

[Several transcription factors participate in the functioning of the alpha-fetoprotein gene promoter]. / Plusieurs facteurs de transcription participent au fonctionnement du promoteur du gène alpha-foetoprotéine.

The oncodevelopmentally regulated alpha-fetoprotein (AFP) gene offers a very good model system to better understand the molecular mechanisms which dictate the specificity of gene expression in liver and control its tight modulation in the course of development and carcinogenesis. Transcription factors of the CCAAT/enhance-binding protein (C/EBP), hepatocyte nuclear factor-1 (HNF-1), and nuclear factor-1 (NF-1) families can bind in vitro to the promoter of the rat AFP gene, which makes the expression of the AFP gene specific to the liver. We have evaluated the influence of some of these factors on the activity of the AFP promoter by transfection of HepG2 hepatoma cells with the appropriate expression vector plus a CAT plasmid under the control of the AFP promoter. A similar plasmid bearing the rat albumin promoter was used as a control. C/EBP alpha, and C/EBP beta acted as transactivators on the AFP promoter, while LIP, a truncated form of C/EBP beta, was a potent negative regulator of the promoter. Interestingly, HNF-1 beta was found to be more potent than HNF-1 alpha in activating the AFP promoter in the HepG2 cells. This effect was highly promoter and cell specific since it did not occur with the rat albumin promoter or in Chinese hamster ovary cells. HNF-1 beta, which is produced earlier than HNF-1 alpha during liver development, would thus have the greater influence on the AFP promoter in early development. Our results pointed to a key role that NF1 might play in the functioning of the AFP promoter. Indeed, overexpression of NF1 induced a specific decrease in the activity of the AFP promoter. Competition between NF1 and HNF-1 for binding to their overlapping binding sites on the AFP promoter would be critical for modulating its activity.

The c-jun proto-oncogene down-regulates the rat alpha-fetoprotein promoter in HepG2 hepatoma cells without binding to DNA.

The effects of a phorbol ester (TPA) and of members of the Jun and Fos oncoprotein family on the activity of the rat alpha-fetoprotein (AFP) promoter were checked by using transient expression experiments in HepG2 hepatoma cells. TPA blocked the activity of the rat AFP promoter in a dose-dependent manner. Overexpression of c-Jun specifically repressed the rat AFP promoter but not the albumin promoter. JunB and JunD were poorer inhibitors. c-Fos expression did not potentiate the negative effect of Jun. The Jun-induced repression does not require binding of c-Jun to the AFP promoter. DNase 1 footprinting experiments did not display any high affinity binding site for Jun on the AFP promoter. Integrity of the c-Jun DNA binding domain is not required for the c-Jun protein to block the AFP promoter. The N-terminal part of Jun, which contains the activating domain, is responsible for the repression as shown by using Jun-Gal4 chimera. Jun likely exerts its negative control on the AFP promoter via protein-protein interactions with a not yet identified trans-activating factor within the -134 to +6 region or with a component of the general machinery of transcription. Jun proteins can thus be key intermediates in regulatory cascades which result in the differential modulation of the AFP and albumin gene expression in the course of liver development and carcinogenesis.

Transformation of methylotrophic yeast Hansenula polymorpha: cloning and expression of genes.

We developed a host-vector system for transformation and gene cloning experiments using the methylotrophic yeast Hansenula polymorpha. Regeneration of protoplasts in a medium containing polyethylene glycol before plating made transformation more efficient and reproducible (2 to 3 x 10(4) micrograms DNA). The frequency of transformation was significantly lower when dominant resistance marker Cup1r was used for transformant selection. The transformation system developed was used to clone the DNA fragment which complements functionally the defect in the dihydroxyacetone kinase (DHAK*) activity of a H. polymorpha mutant strain. The DNA insert isolated was shown to increase by up to ten times the activity of DHAK in transformants carrying recombinant plasmids. When recombinant plasmids were introduced into S. cerevisiae, the transformants obtained acquired the ability to grow on the medium with dihydroxyacetone as a sole carbon source and the activity of DHAK was observed.

Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha.

For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.

Ribosomal RNA synthesis in liver of adrenalectomized rats after partial hepatectomy.

The ribosome formation in four experimental groups: normal, adrenalectomized, partially hepatectomized and adrenalectomized - partially hepatectomized rats was studied. Ribosomal RNA was labelled for different intervals and the transfer of the radioactivity from 45 S pre-rRNA through the nucleolar pre-rRNA and rRNA pools into cytoplasmic 28S and 18S rRNA was followed. The results show that there are at least two ways of positive control of rRNA synthesis, one of them being glucocorticoid-dependent. The acceleration of the pre-rRNA processing through the shortest maturation pathway in regenerating liver is reduced in the absence of the hormone. Glucocorticoids do not influence nucleo-cytoplasmic rRNA transport.

Acceleration of ribosome formation in rat liver in response to hydrocortisone.

The amount of nuclear pre-rRNA (precursor to rRNA) and rRNA species and their labelling in vivo with [14C]orotate were determined in hormone-deficient and treated rats. On the basis of the data obtained, the rates of synthesis and processing of pre-rRNA in the two groups of animals were compared. It is shown that the rate of nucleolar RNA synthesis is increased twice, its half-life is reduced about 1.4 times and the endonuclease processing of primary pre-rRNA is channeled through the shortest maturation pathway. Thus, an accelerated ribosome formation takes place in response to hydrocortisone. The overproduced ribosomes are not retained in the nucleoplasm and are immediately transferred to the cytoplasm.