Maximilian Binder

A C-terminal region of the Saccharomyces cerevisiae transcription factor ADR1 plays an important role in the regulation of peroxisome proliferation by fatty acids

The Saccharomyces cerevisiae transcriptional activator ADR1, which controls ADH2 gene expression, was shown to be involved in the regulation of peroxisome proliferation. To study the mode of action of ADR1, we compared strains carrying the adr1-1 mutation, high or low copy numbers of the ADR1 gene, the constitutive allele ADR1-5c, and 3′-deletions of ADR1. High ADR1 gene dosage increased the transcription of genes encoding peroxisomal proteins as compared to one copy of the ADR1 gene. Furthermore, overexpression of ADR1 under ethanol growth conditions induced the proliferation of peroxisomal structures. The organelles were observed to be localized in clusters, a typical feature of peroxisomes induced by oleic acid. In contrast, the ADR1-5c allele, which induces ADH2 expression to a level comparable to that of high ADR1 gene dosage was found to have only a small effect. An analysis of functional domains of the ADR1 protein revealed that the N-terminal 220 amino acids of ADR1 were sufficient for wild-type levels of transcription of the FOX2, FOX3, and PAS1 genes, but the entire ADR1 protein was required for complete induction of the CTA1 gene and for growth oleic acid medium. Our data suggest that a functional domain of the ADR1 protein localized between residues 643 and 1323 is required for the induction of peroxisomal structures and for the utilization of oleic acid.

Regulation of the yeast SPS19 gene encoding peroxisomal 2,4‐dienoyl‐CoA reductase by the transcription factors Pip2p and Oaf1p: β‐oxidation is dispensable for Saccharomyces cerevisiae sporulation in acetate medium

The yeast SPS19 gene encoding the peroxisomally targeted 2,4‐dienoyl‐CoA reductase shares its promoter region (291 bp) with the sporulation‐specific gene SPS18. SPS19 is induced during sporulation in diploids but to a lesser extent than SPS18; under oleate induction conditions, SPS19, but not SPS18, is transcribed via an oleate response element (ORE) independently of ploidy or sporulation. The SPS19 ORE is the binding target of the Pip2p and Oaf1p transcription factors, and an SPS19–lacZ reporter gene, which is highly expressed in oleate‐induced cells, is not activated in haploids devoid of either protein. We examined the expression of CYC1–lacZ reporter constructs carrying the SPS19 and CTA1 OREs in diploids propagated under sporulation conditions and have shown that OREs are not sufficient for heterologous expression during yeast development. In addition, diploids deleted at either PIP2 or OAF1 demonstrated abundant ascosporogenesis, indicating that these genes are not essential for sporulation. A Δpex6 strain lacking peroxisomal structures and one devoid of fatty acyl‐CoA oxidase (Δpox1), the first step in fungal β‐oxidation, were both proficient for sporulation and, hence, β‐oxidation and the peroxisomal compartment containing it are dispensable for meiotic development.

Oestradiol-BSA conjugates for receptor histochemistry: problems of stability and interactions with cytosol

SummaryThe validity of histochemical methods for the localization of hormone receptors based on the binding of fluorescent bovine serum albumin conjugates of oestradiol was examined with respect to their stability and their interactions with the oestrogen receptor type I. Stability was assessed by measuring free oestrogen in conjugates by radioimmunoassay and/or receptor protein binding assay. Sufficient free oestrogen-in order to saturate type I and type II binding sites (ER I, ER II)-was detected in freshly prepared conjugates. This free oestrogen originates in inadequate removal of adsorptively bound original ligand after synthesis. Apart from this fact, conjugates appeared to be unstable in aqueous solutions, especially under the conditions used for histochemical methods. Free oestrogen extracted from the conjugates was subjected to high performance liquid chromatography. Amongst the eluted peaks, oestradiol and/or the original ligand used for synthesis were identified. Thein vitro interaction of conjugates with oestrogen receptors was studied by competitive binding analysis and by incubation of cytosol with a Sepharose-bound conjugate. The results, especially those concerning the amount of free oestrogen, suggest that neither ER I nor ER II is involved in the staining mechanism of conjugates.

Cytogenetic demonstration of mitotic chromosomes in the yeast Saccharomyces cerevisiae.

SummaryA cytogenetic study of the nuclear material of the yeast Saccharomyces cerevisiae as it appears during mitosis was undertaken using a technique which combines light and electron microscopic examination of spheroplasts. Condensation of chromatin into separate chromosomes is observed. The most frequent chromosome number counted is 18.

Fate and role of peroxisomes during the life cycle of the yeast Saccharomyces cerevisiae: inheritance of peroxisomes during meiosis

Abstract Sporulation in the yeast Saccharomyces cerevisiae is a meiotic developmental process that occurs in MATa/MATα heterozygotes in response to nutrient deprivation. Here, the fate and role of peroxisomes during sporulation and germination has been examined by a combination of immunoelectron microscopy and the use of pex mutants defective in peroxisomal functions. Using a green fluorescent protein probe targeted to peroxisomes we show that peroxisomes are inherited through meiosis and that they do not increase in number either during sporulation or spore germination. In addition, there is no requirement for peroxisome degradation prior to spore packaging. Unlike the situation in filamentous fungi, peroxisomes do not proliferate during the yeast life cycle. Functional peroxisomes are dispensable for efficient meiotic development on acetate medium since homozygous Δpex6 diploids sporulated well and produced mature spores that were resistant to diethyl ether. Like haploids, diploid cells can proliferate their peroxisomes in response to oleate as sole carbon source in liquid medium, but under these conditions they do not sporulate. On solid oleate medium, homozygous pex5,Δpex6, and pex7 cells were unable to sporulate efficiently, whereas the wild type was. The results presented here are discussed in terms of the transmission of organelles to progeny cells.

Immunogold labeling of yeast cells: an efficient tool for the study of protein targeting and morphological alterations due to overexpression and inactivation of genes.

Immunogold labeling on Lowicryl HM20 resin sections is a valuable complement to biochemical methods as well as methods of molecular biology in the study of basic mechanisms in the yeast system. This contribution presents an overview of the state of the art. Emphasis is put on the explanation of caveats and pitfalls rather than on detailed bench protocols. In the Applications section the morphological aspect of genetic manipulation is accentuated and links to human pathology are indicated. The morphological consequences of genetic manipulations may gain importance in view of the efforts made to establish gene therapies. In particular, the contribution of immunoelectron microscopy to the elucidation of peroxisomal targeting signals and to the detection and identification of morphological alteration due to overexpressed, mutated or deleted genes in the context of peroxisome biogenesis is described.

A study of the chromosomes of the yeast Schizosachcharomyces pombe by light and electron microscopy

Summary A cytogenetic analysis of the yeast, Schizosaccharomyces pombe , by light and electron microscopy is presented. An examination of identical figures by both methods demonstrates that the resolution of the light microscope does not allow an accurate count of the chromosomes, whose size varies from 0.1 to 0.6 μm. From 104 spreads, examined by electron microscopy, 35 with light microscope correlations, a haploid chromosome number of 8 is suggested for this yeast.