Nucleosome Remodeling at the Yeast and Promoters without the Putatively Essential SWI/SNF Remodeler.

Chromatin remodeling by ATP-dependent remodeling enzymes is crucial for all genomic processes, like transcription or replication. Eukaryotes harbor many remodeler types, and it is unclear why a given chromatin transition requires more or less stringently one or several remodelers. As a classical example, removal of budding yeast and promoter nucleosomes upon physiological gene induction by phosphate starvation essentially requires the SWI/SNF remodeling complex.

Antisense non-coding transcription represses the PHO5 model gene at the level of promoter chromatin structure.

Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. A negative role for antisense transcription at the PHO5 gene locus was demonstrated by leveraging the level of overlapping antisense transcription through specific mutant backgrounds, expression from a strong promoter in cis, and use of the CRISPRi system.

The yeast PHO5 promoter: from single locus to systems biology of a paradigm for gene regulation through chromatin.

Chromatin dynamics crucially contributes to gene regulation. Studies of the yeast PHO5 promoter were key to establish this nowadays accepted view and continuously provide mechanistic insight in chromatin remodeling and promoter regulation, both on single locus as well as on systems level. The PHO5 promoter is a context independent chromatin switch module where in the repressed state positioned nucleosomes occlude transcription factor sites such that nucleosome remodeling is a prerequisite for and not consequence of induced gene transcription.

The RSC chromatin remodeling complex has a crucial role in the complete remodeler set for yeast PHO5 promoter opening.

Although yeast PHO5 promoter chromatin opening is a founding model for chromatin remodeling, the complete set of involved remodelers remained unknown for a long time. The SWI/SNF and INO80 remodelers cooperate here, but nonessentially, and none of the many tested single or combined remodeler gene mutations could prevent PHO5 promoter opening. RSC, the most abundant and only remodeler essential for viability, was a controversial candidate for the unrecognized remodeling activity but unassessed in vivo.

Differential cofactor requirements for histone eviction from two nucleosomes at the yeast PHO84 promoter are determined by intrinsic nucleosome stability.

We showed previously that the strong PHO5 promoter is less dependent on chromatin cofactors than the weaker coregulated PHO8 promoter. In this study we asked if chromatin remodeling at the even stronger PHO84 promoter was correspondingly less cofactor dependent. The repressed PHO84 promoter showed a short hypersensitive region that was flanked upstream and downstream by a positioned nucleosome and contained two transactivator Pho4 sites.

Redundancy of chromatin remodeling pathways for the induction of the yeast PHO5 promoter in vivo.

Induction of the yeast PHO5 and PHO8 genes leads to a prominent chromatin transition at their promoter regions as a prerequisite for transcription activation. Although induction of PHO8 is strictly dependent on Snf2 and Gcn5, there is no chromatin remodeler identified so far that would be essential for the opening of PHO5 promoter chromatin. Nonetheless, the nonessential but significant involvement of cofactors can be identified if the chromatin opening kinetics are delayed in the respective mutants.

The histone chaperone Asf1 increases the rate of histone eviction at the yeast PHO5 and PHO8 promoters.

Eukaryotic gene expression starts off from a largely obstructive chromatin substrate that has to be rendered accessible by regulated mechanisms of chromatin remodeling. The yeast PHO5 promoter is a well known example for the contribution of positioned nucleosomes to gene repression and for extensive chromatin remodeling in the course of gene induction. Recently, the mechanism of this remodeling process was shown to lead to the disassembly of promoter nucleosomes and the eviction of the constituent histones in trans.

Multiple mechanistically distinct functions of SAGA at the PHO5 promoter.

Our previous studies have shown that the rate of chromatin remodeling and consequently the rate of PHO5 activation are strongly decreased in the absence of Gcn5 histone acetyltransferase activity. Using chromatin immunoprecipitation, we demonstrate that SAGA is physically recruited to the PHO5 promoter. Recruitment is dependent on the specific activator Pho4 and occurs only under inducing conditions.

Increasing the rate of chromatin remodeling and gene activation--a novel role for the histone acetyltransferase Gcn5.

Histone acetyltransferases (HATs) such as Gcn5 play a role in transcriptional activation. However, the majority of constitutive genes show no requirement for GCN5, and even regulated genes, such as the yeast PHO5 gene, do not seem to be affected significantly by its absence under normal activation conditions. Here we show that even though the steady-state level of activated PHO5 transcription is not affected by deletion of GCN5, the rate of activation following phosphate starvation is significantly decreased.

Transcriptional regulation of the yeast PHO8 promoter in comparison to the coregulated PHO5 promoter.

Expression of the PHO8 and PHO5 genes that encode a nonspecific alkaline and acid phosphatase, respectively, is regulated in response to the P(i) concentration in the medium by the same transcription factors. Upon induction by phosphate starvation, both promoters undergo characteristic chromatin remodeling, yet the extent of remodeling at the PHO8 promoter is significantly lower than at PHO5. Despite the coordinate regulation of the two promoters, the PHO8 promoter is almost 10 times weaker than PHO5.

[Importance of assessment of visual function in nautical school students and naval engineers].

Eighty-seven junior nautical school students, of which 46 undergraduate marine engineers and 41 undergraduate nautical officer were examined for visual functions as a part of screening procedure for nautical occupations. All students were male, aged between 17 and 18. Examination comprised visual acuity, visual depth, and colour vision.

Analyzing chromatin structure and transcription factor binding in yeast.

The study of chromatin, once thought to be a purely structural matrix serving to compact the DNA of the genome into the nucleus, is of increasing value for our understanding of how DNA functions in the cell. This article provides two basic procedures for the study of chromatin in vivo. The first is a DNase I-based method for the treatment of isolated nuclei to resolve the chromatin structure of a particular region; the second employs dimethyl sulfate footprinting of whole cells in vivo to determine the binding of factors to cis elements in the locus of interest.

Cooperative Pho2-Pho4 interactions at the PHO5 promoter are critical for binding of Pho4 to UASp1 and for efficient transactivation by Pho4 at UASp2.

The activation of the PHO5 gene in Saccharomyces cerevisiae in response to phosphate starvation critically depends on two transcriptional activators, the basic helix-loop-helix protein Pho4 and the homeodomain protein Pho2. Pho4 acts through two essential binding sites corresponding to the regulatory elements UASp1 and UASp2. Mutation of either of them results in a 10-fold decrease in promoter activity, and mutation of both sites renders the promoter totally uninducible.

The homeodomain protein Pho2 and the basic-helix-loop-helix protein Pho4 bind DNA cooperatively at the yeast PHO5 promoter.

Two transcription factors, the bHLH protein Pho4 and the homeodomain protein Pho2, are required for transcriptional activation of the PHO5 promoter in Saccharomyces cerevisiae. There are two essential Pho4 binding sites, corresponding to the regulatory elements UASp1 and UASp2 at the PHO5 promoter, but only a single, dispensable Pho2 binding site had previously been identified. We have reinvestigated binding of Pho2 to the PHO5 promoter using purified recombinant protein and have found multiple Pho2 binding sites of different affinities along the promoter.

[Occupational medicine in health care legislation with special emphasis on organizational possibilities].

Primary health care in Croatia, and preventive health care in particular, are organized in compliance with international conventions. In the Croatian Health Care Act special attention is paid to the worker's health. The responsibility for implementing specific health care measures lies with the employer who has the duty to care for the health and safety of his employees.

[The importance of a medical evaluation in adolescence within the field of vocational guidance].

An investigation was carried out among the rural adolescent population in the Zadar region. The objective was to examine their health status, to gain information about occupations they wish to be trained for and to find out whether there are any medical contraindications in respect to those occupations. Using methods of vocational guidance medicine health disorders were found to be present in 30% of the examined adolescents.

Specific dephosphorylation of phosphopeptides by the yeast alkaline phosphatase encoded by PHO8 gene.

Partially purified nonspecific phosphate-repressible alkaline phosphatase from Saccharomyces cerevisiae encoded by PHO8 gene (rALPase), efficiently dephosphorylates phosphohistones and a variety of phosphopeptides. The pho8 mutant, constructed by disruption of the chromosomal counterpart of the PHO8 gene, is lacking in phosphatase activity toward phosphopeptides, confirming that this activity is actually due to rALPase. rALPase activity tested on phosphopeptides is maximum in the pH range 6.

Binding of Saccharomyces cerevisiae extracellular proteins to glucane.

Interactions of Saccharomyces cerevisiae cell wall proteins with purified yeast glucane were studied. Using the beta-glucanase (BGL2 gene product) as the model cell wall protein, strong binding to glucane was demonstrated at pH lower than 7, while at pH higher than 8 the reaction did not occur. NaCl (2 M) did not influence the binding, while urea in concentrations higher than 4 M affected the interactions.

Activation of the weakly regulated PHO8 promoter in S. cerevisiae: chromatin transition and binding sites for the positive regulatory protein PHO4.

PHO8 encodes an alkaline phosphatase in Saccharomyces cerevisiae whose transcription is regulated by the phosphate concentration in the medium. This occurs through the action of several positive and negative regulatory proteins, also involved in the regulation of other members of the phosphatase gene family. A central role is played by PHO4, the gene encoding a DNA binding regulatory protein.

Expression, glycosylation and secretion of yeast acid phosphatase in hamster BHK cells.

The gene PHO5 coding for one of the repressible acid phosphatases of the yeast Saccharomyces cerevisiae has been expressed at high efficiency in the baby hamster kidney (BHK) cell line. The expression vector was constructed from PHO5 driven by the human beta-actin promoter and was transfected into BHK cells by the calcium phosphate method. The recombinant APase (r-APase) which was secreted in active form from the cells was estimated by SDS/polyacrylamide gel electrophoresis to have molecular mass M(r) = 62,000, indicating substitution of the polypeptide moiety by 2-3 asparagine-linked glycans.

Influence of glycosylation on the oligomeric structure of yeast acid phosphatase.

Secreted yeast acid phosphatase is found to be an octamer under physiological conditions rather than a dimer, as previously believed. The octameric form of the enzyme dissociates rapidly into dimers at pH below 3 and above 5, or by treatment with guanidine hydrochloride or urea, without further dissociation of dimers. Crosslinking experiments revealed that the dissociation of the octamer occurs through very unstable hexamers and tetramers, showing that the octamer is built of dimeric units.

Stabilization of glycoenzymes by cross-linking of their carbohydrate chains.

The results presented here demonstrate the potential applicability of the described cross-linking method for preparation of soluble glycoenzyme derivatives. The high level of the retained enzyme activity supports the assumption that this approach is superior to the cross-linking through the protein part. In this study, high mannose-type glycoproteins were used.

Preparation of the stabilized glycoenzymes by cross-linking their carbohydrate chains.

Each of the three high-mannose type glycoproteins studied, acid phosphatase, invertase, and glucose oxidase, could be specifically cross-linked through its carbohydrate chains. The procedure involves periodate oxidation of carbohydrate residues followed by reaction of the generated aldehyde groups with adipic acid dihydrazide as a cross-linker. The amount and size as well as solubility of the formed polymers could be efficiently controlled by varying the reaction conditions, i.

Role of glycosylation in secretion of yeast acid phosphatase.

The minimal glycosylation requirement for acid phosphatase secretion and activity was investigated using tunicamycin, an inhibitor of protein glycosylation, and a yeast mutant defective in the synthesis of oligosaccharide outer chains. The results obtained show that outer chain addition is not essential for secretion of active enzyme and that only 4 core chains, out of 8 normally attached to a protein subunit, are sufficient for enzyme transport to the periplasmic space. Enzyme forms with less than 4 chains were retained in membranes of endoplasmic reticulum.