Molecular cloning and characterization of the Saccharomyces cerevisiae SAB1 gene that suppresses a temperature-sensitive phenotype of the ARS-binding factor 1 mutant.

A high-copy number suppressor gene of the yeast temperature-sensitive lethal abf1 mutant was isolated and named SAB1 (suppressor of ABF1). Chromoblot hybridization and grid-filter hybridization analyses showed that the SAB1 gene was located on chromosome IV. Deletion analyses of the SAB1 plasmid revealed that the suppressor activity was contained in a 1.1 kb DNA region. The nucleotide sequence of the 1.1 kb DNA fragment was determined and turned out to be identical to that of the yeast phosphoribosylanthranilate isomerase gene (TRP1). A binding site for ARS-Binding Factor 1 was located in the coding sequence of the TRP1 gene, which has been known to be a part of the B domain of yeast autonomously replicating sequence 1 (ARS1). Our results suggest that ABF1 might be important for the transcription of the yeast TRP1 gene in addition to having important roles in the stimulation of replication at the ARS1 locus.

Single-chain MHC class II molecules induce T cell activation and apoptosis.

MHC class II/peptide complexes displayed on the surface of APCs play a pivotal role in initiating specific T cell responses. Evidence is presented here that components of this heterotrimeric complex can be genetically linked into a single polypeptide chain. Soluble single-chain (sc) murine class II IA(d) molecules with and without covalently attached peptides were produced in a recombinant baculovirus-insect cell expression system. Correct conformation of these molecules was verified based on 1) reactivity to Abs directed against conformational epitopes in IA(d) and 2) peptide-specific recognition of the IA(d)/peptide complexes by T cells. Both sc class II molecules loaded the appropriate peptides and sc class II/peptide fusions were effective in stimulating T cell responses, including cytokine release and apoptosis. Mammalian cells were also found to be capable of expressing functional sc class II molecules on their cell surfaces. The findings reported here open up the possibility of producing large amounts of stable sc class II/peptide fusion molecules for structural characterization and immunotherapeutic applications.

Isolation and characterization of Saccharomyces cerevisiae SAB2, a suppressor gene for temperature-sensitive phenotype of ARS-binding factor 1 mutant.

A high-copy number suppressor of yeast abf1-5 mutant, a temperature-sensitive lethal mutant, was isolated and named SAB2 (suppressor of ABF1). Hybridization to a yeast chromoblot and to prime clone grid filters revealed that the SAB2 gene was located near the yeast SUP3 on chromosome XV. The suppressor activity was contained in a 2.5 Kbp DNA region of the SAB2 plasmid. The nucleotide sequence of the DNA region contained a long open reading frame, which turned out to encode for yeast tryptophan permease. Four putative ABF1 binding sites were found in the promoter and the structural regions of the tryptophan permease gene. Binding of ABF1 to two of the sites tested in this study was detected. Our results indicate that ABF1 may be involved in the transcriptional control of the yeast tryptophan permease gene.

ABF1 is a phosphoprotein and plays a role in carbon source control of COX6 transcription in Saccharomyces cerevisiae.

Previously, we have shown that the Saccharomyces cerevisiae DNA-binding protein ABF1 exists in at least two different electrophoretic forms (K. S. Sweder, P. R. Rhode, and J. L. Campbell, J. Biol. Chem. 263: 17270-17277, 1988). In this report, we show that these forms represent different states of phosphorylation of ABF1 and that at least four different phosphorylation states can be resolved electrophoretically. The ratios of these states to one another differ according to growth conditions and carbon source. Phosphorylation of ABF1 is therefore a regulated process. In nitrogen-starved cells or in cells grown on nonfermentable carbon sources (e.g., lactate), phosphorylated forms predominate, while in cells grown on fermentable carbon sources (e.g., glucose), dephosphorylated forms are enriched. The phosphorylation pattern is affected by mutations in the SNF1-SSN6 pathway, which is involved in glucose repression-depression. Whereas a functional SNF1 gene, which encodes a protein kinase, is not required for the phosphorylation of ABF1, a functional SSN6 gene is required for itsd ephosphorylation. The phosphorylation patterns that we have observed correlate with the regulation of a specific target gene, COX6, which encodes subunit VI of cytochrome c oxidase. Transcription of COX6 is repressed by growth in medium containing a fermentable carbon source and is derepressed by growth in medium containing a nonfermentable carbon source. COX6 repression-derepression is under the control of the SNF1-SSN6 pathway. This carbon source regulation is exerted through domain 1, a region of the upstream activation sequence UAS6 that binds ABF1 (J. D. Trawick, N. Kraut, F. Simon, and R. O. Poyton, Mol. Cell Biol. 12:2302-2314, 1992). We show that the greater the phosphorylation of ABF1, the greater the transcription of COX6. Furthermore, the ABF1-containing protein-DNA complexes formed at domain 1 differ according to the phosphorylation state of ABF1 and the carbon source on which the cells were grown. From these findings, we propose that the phosphorylation of ABF1 is involved in glucose repression-derepression of COX6 transcription.

Role of multifunctional autonomously replicating sequence binding factor 1 in the initiation of DNA replication and transcriptional control in Saccharomyces cerevisiae.

Autonomously replicating sequence (ARS) binding factor 1 (ABF1) is an abundant DNA-binding protein that specifically recognizes the motif RTCRYN5ACG at many sites in the yeast genome, including promoter elements, mating-type silencers, and ARSs. Mutational analysis of these sites suggests that ABF1 is involved in constitutive and carbon source-regulated transcriptional activation, transcriptional silencing, and ARS activity. To better assess the role of ABF1 in DNA replication and transcriptional control, temperature-sensitive lethal mutations in the ABF1 gene were isolated. Several of the abf1(Ts) strains show rapid growth arrest at the nonpermissive temperature. At the semipermissive temperature, these strains show an ARS-specific defect in the mitotic stability of ARS-CEN plasmids, such that the abf1 mutants show defects in ARS function identical to those of mutants bearing the mutations in the cis-acting ABF1 binding sites analyzed previously by numerous investigators. Flow cytometric analysis and in vivo DNA labeling experiments on an alpha-factor synchronized abf1(Ts) strain showed that at the nonpermissive temperature, these cells fail to progress efficiently from G1 through S phase and synthesize DNA at 25% of the level seen in the isogenic ABF1 strain. RNA synthesis is also reduced in the abf1(Ts) strains. In addition, transcriptional activation by an ABF1 binding site upstream activation sequence is completely defective in an abf1(Ts) strain at the semipermissive temperature. These phenotypes provide evidence that the same protein, ABF1, functions in the initiation of DNA replication and transcriptional activation.

Inhibitory effects of serum and stimulatory effects of estrogen on prolactin mRNA levels in GH3 rat pituitary tumor cells.

We investigated the effects of serum and estrogen on prolactin (PRL) mRNA accumulation in GH3 cells under different cell culture conditions. Hybridization analysis of GH3 cellular RNA indicated that PRL mRNA levels decreased more than 20-fold in cells cultured for 1 week in medium containing dextran-charcoal-treated serum (stripped serum). No effects on actin mRNA levels were observed under these conditions. Furthermore, this inhibition of PRL mRNA accumulation depended on the concentration of stripped serum in the medium. Although incubation in stripped-serum medium inhibited cell growth, these culture conditions did not appear to irreversibly affect the GH3 cell population. These data indicate that a potent inhibitor of PRL mRNA accumulation is present in stripped serum. GH3 cells grown in stripped-serum medium were shown to be responsive to estrogen. Treatment of these cells with 10(-9) M estradiol resulted in a 6.6-fold stimulation of PRL gene expression. However, estrogen had no effect on cell growth under these conditions, suggesting that estrogen stimulates PRL gene expression and cell proliferation by independent mechanisms.

Growth and cell cycle regulation of mRNA levels in GH3 cells.

In the rat anterior pituitary gland, estrogen increases both prolactin (PRL) mRNA levels and stimulates the proliferation of PRL-producing cells. The temporal sequence of these events suggests that PRL gene expression may be coordinated with cell proliferation. We investigated the relationship between cell cycle progression and the accumulation of the PRL mRNA, as well as several other mRNAs, in the rat pituitary tumor GH3 cell line. Serum-deprived cells progressed from G0 to S phase in 20-24 h following serum stimulation. During this time, beta-actin mRNA levels increased 7-fold in 5 h, then returned to basal levels prior to the beginning of S phase. Histone H1 mRNA levels increased approximately 3-fold as cells entered S phase. These data are consistent with the cell cycle-dependent regulation of beta-actin and histone H1 gene expression reported for other cell types. Glucocorticoid receptor mRNA levels were barely detectable in serum-deprived cells but rapidly increased 3- to 5-fold following serum stimulation. This increase resulted in glucocorticoid receptor mRNA levels that were equivalent to those seen in cells maintained in serum-containing medium, suggesting that serum factors regulate glucocorticoid receptor gene expression. In contrast to these changes in gene expression, the levels of PRL and growth hormone (GH) mRNAs gradually increased 2-fold while the cells progressed through G1 phase. Similarly, in cells synchronized to progress through S and G2 phases following aphidicolin treatment, histone H1 gene expression showed a specific increase in S phase cells, whereas PRL and GH mRNA levels changed little with cell cycle progression. These results indicate that the levels of PRL and GH mRNAs are not regulated in a cell cycle-dependent manner. When changes in estrogen responsiveness were determined during the cell cycle, we found that estradiol treatment was capable of increasing PRL mRNA accumulation independent of cell cycle progression and cell cycle distribution in synchronized GH3 cells. These results support the hypothesis that the hormonal regulation of PRL gene expression is not significantly affected by cell growth.

The gene encoding ARS-binding factor I is essential for the viability of yeast.

The gene encoding a yeast ARS-binding protein, ABF I, has been cloned by screening a genomic lambda gt11 library using monoclonal and polyclonal antibodies against ABF I. ABF I is of interest because it not only binds to ARSs but also to the 5'-flanking region of genes encoding proteins involved in transcription, translation, respiration, and cell-cycle control. The cloned gene has been used to prepare null mutants, which further demonstrate the importance of the ABF I protein by showing that it is essential for vegetative growth. ABF1 maps to chromosome V. The DNA sequence of the ABF1 gene reveals several motifs characteristic of DNA-binding proteins but shows no overall similarity to any protein of known function.

Purification and characterization of proteins that bind to yeast ARSs.

Two proteins that bind to yeast ARS DNA have been purified using conventional and oligonucleotide affinity chromatography. One protein has been purified to homogeneity and has a mass of 135 kDa. Competitive binding studies and DNase I footprinting show that the protein binds to a sequence about 80 base pairs away from the core consensus in the region known as domain B. This region has previously been shown to be required for efficient replication of plasmids carrying ARS1 elements. To investigate further whether the protein might have a function related to the ability of ARSs to act as replicators, binding to another ARS was tested. The protein binds to the functional ARS adjacent to the silent mating type locus HMR, called the HMR-E ARS, about 60 base pairs from the core consensus sequence. Surprisingly, there is little homology between the binding site at the HMR-E ARS and the binding site at ARS1. The 135-kDa protein is probably the same as ABF-I (SBF I) (Shore, D., Stillman, D. J. Brand, A. H., and Nasmyth, K. A. (1987) EMBO J. 6, 461-467; Buchman, A. R., Kimmerly, W. J., Rine, J., and Kornberg, R. D. (1988) Mol. Cell. Biol. 8, 210-225). A second DNA-binding protein was separated from ABF-I during later stages of the purification. This protein, which we designate ABF-III, also binds specifically to the ARS1 sequence, as shown by DNase I footprinting, at a site adjacent to the ABF-I recognition site. Purification of these two ARS binding proteins should aid in our understanding of the complex mechanisms that regulate eukaryotic DNA replication.