Fermentable and nonfermentable carbon sources sustain constitutive levels of expression of yeast triosephosphate dehydrogenase 3 gene from distinct promoter elements.

The triosephosphate dehydrogenase 3 gene (TDH3) is a glycolytic enzyme gene and is abundantly transcribed in Saccharomyces cerevisiae. The promoter region of the TDH3 gene is known to exhibit high transcriptional activity regardless of the fermentability of the carbon source and has been widely utilized to synthesize heterologous gene products in S. cerevisiae. To clarify the mechanism of constitutive transcription by the promoter, we constructed mutant promoters and analyzed the in vivo transcriptional activity of these promoters. The majority of the transcriptional potential is contained within a DNA fragment extending from nucleotides -524 to -255 (-524/-255; relative to the translation initiation codon), which consists of three cis-acting elements: a fermentable carbon source-dependent upstream activation sequence (UAS) 1 (-524/-426), a fermentable carbon source-dependent upstream repression sequence (URS) (-426/-393), and a nonfermetable carbon source-dependent UAS2 (-305/-255). This result indicates that the promoter involves two apparent promoter elements. One is fermentable carbon source-dependent, and another is nonfermentable carbon source-dependent. Southwestern analyses indicated that a novel 20-kDa protein is induced in yeast cells by shifting from a fermentable to nonfermentable carbon source. The protein interacts with two UAS1 13-base pair elements and one URS 13-base pair element, one of which had been previously designated GPE (general regulatory factor 1 (GRF1) binding site potentiator element) (Bitter, G. A., Chang, K. K. H., and Egan, K. M. (1991) Mol. Gen. Genet. 231, 22-32). We therefore termed the 20-kDa protein GPEB (GRF1-binding site potentiator element-binding protein). In addition, mutational analyses strongly suggested that UAS1, URS, and UAS2 interact with GRF1 and GPEB, GPEB, and the GCR1 (glycolysis regulation 1) gene product, respectively. We therefore concluded that constitutive transcription by the TDH3 promoter is sustained by two promoter elements and that the switch between them might be controlled by the nonfermentable carbon source-inducible GPEB.

Saccharomyces cerevisiae can release hepatitis B virus surface antigen (HBsAg) particles into the medium by its secretory apparatus.

We constructed a plasmid that directs the synthesis and secretion of hepatitis B virus (HBV) surface antigen (HBsAg) particles by Saccharomyces cerevisiae. This plasmid contains a proteinase-resistant HBsAg M (M-P31c) gene fused at its 5'-terminus with a chicken-lysozyme signal peptide (C-SIG) gene, which is placed under the yeast GLD (glyceraldehyde-3-phosphate dehydrogenase gene) promoter. The products encoded by the "C-SIG+M-P31c" (LM-P31c) gene were synthesized and assembled themselves into HBsAg particles in yeast cells, and the particles were released into the medium along with poly-HSA (polymerized human serum albumin) binding activity. The HBsAg particles purified from the medium were very similar in density (1.19 g cm-3), size (19.2 +/- 0.8 nm in diameter) and shape (sphere) to human-plasma-derived HBsAg particles. When several sec (temperature-sensitive secretion-defective) mutants were used as host cells, the release of HBsAg particles into the medium was blocked at 37 degrees C but not at 25 degrees C, indicating that the HBsAg particles are exported through the normal yeast secretion pathway. To our knowledge, this is the first report that yeast cells are capable of secreting particles into the medium.

Identification of polymerized-albumin receptor domain in the pre-S2 region of hepatitis B virus surface antigen M protein.

The pre-S2-coding region in the hepatitis B virus surface antigen M (P31; pre-S2 + S) protein gene was modified to identify a polymerized-albumin receptor (PAR) domain by deleting restriction fragments or performing site-directed mutagenesis. The modified M protein genes (M-P31x; x = d, e, f, h and i) were cloned into the yeast generalized-expression vector pGLD 906-1 and expressed in Saccharomyces cerevisiae under the control of yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. The PAR activities of these gene products suggested that the PAR domain is located in the hydrophilic and highly conserved domain in the pre-S2 region (around Leu12 approximately Tyr21). Antibodies specific for a pre-S2 peptide (Phe8 approximately Pro34, subtype adr), which covers the PAR domain, were purified from sera of rabbits immunized with yeast-derived M protein particles having a natural PAR domain. Immune electron microscopy showed that the purified antibodies could aggregate HBV particles. Therefore, it was speculated that the PAR domain overlapped with the dominant virus-neutralizing and virus-protecting epitopes.

Hepatitis B virus envelope L protein particles. Synthesis and assembly in Saccharomyces cerevisiae, purification and characterization.

The hepatitis B virus envelope gene encodes three transmembrane proteins in frame; S, the product of S gene; M, the product of M (pre-S2 + S) gene; and L, the product of L (pre-S1 + pre-S2 + S) gene. Unlike the S and M proteins, attempts to efficiently synthesize L proteins and assemble them into L protein particles in various eukaryotic cells have been unsuccessful, probably because of the presence of the pre-S1 peptide with an unknown function which appears to be inhibitory to the host secretory apparatus. To investigate the role of the pre-S1 peptide, we constructed an L gene fused with a synthetic gene for chicken-lysozyme signal peptide (C-SIG) at the 5'-terminal and placed the resultant gene under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. After the fused-C-SIG peptide was correctly processed by the yeast secretory apparatus, a yeast transformant synthesized a protein with a molecular mass of approximately 52 kDa at a level of 42% of the total soluble protein. Electron micrographic observation showed that the gene products assembled into 23-nm spherical and filamentous particles. The pre-S peptide of the gene product was deposited into the endoplasmic reticulum (ER) lumen and well-glycosylated. It seemed that the gene products were accumulated as particles in certain specific membrane structures of the yeast secretory apparatus. Moreover, both the amount of mRNAs specific for the L gene and the in vivo stability of the synthesized L proteins did not change significantly by the addition of the C-SIG gene. These findings indicated that, if the pre-S1 peptide penetrates the ER membrane efficiently, the L proteins can be synthesized cotranslationally, translocate across the ER membrane with its S region, and then assemble by themselves into the particle form. Therefore, the pre-S1 peptide may involve weak or reduced signal peptide activity for recognition by the secretory apparatus and/or for the transport of the pre-S peptide into the ER lumen.

[Improvement of pituitary homograft under the kidney capsule in mice].

The model of hyperprolactinemia induced by pituitary homografts under the kidney capsule has been used mainly in the field of reproductive physiology. The authors report an improved method for pituitary grafting in mice. The procedure was as follows: 1. The male pituitary glands with normal saline were aspirated into a polyethylene tube. 2. Two incisions were made in the kidney capsule. 3. The polyethylene tube with pituitary glands was inserted via a large incision. 4. Blowing air into the tube, the pituitary glands were left under the kidney capsule and normal saline streamed out of a small incision. Using this method, all pituitary grafted mice became pseudopregnant.