[Long-term results of sentinel node biopsy diagnostics in penile carcinoma : Dynamic sentinel node biopsy in cases with nonpalpable lymph nodes in the groin]. / Langzeitergebnisse der Sentinel-Lymphknotendiagnostik beim Peniskarzinom : Dynamische Sentinel-Lymphknotenbiopsie bei nicht-palpablen Leistenlymphknoten.

OBJECTIVE: Dynamic sentinel node biopsy (DSNB) has been recommended in the EAU guidelines for several years as a minimally invasive method for lymph node staging in patients with penile carcinoma and nonpalpable lymph nodes. However, due to the high methodological demands and the primarily unreliable results, this method is rarely used in Germany. The aim of this study was to establish the reliability and morbidity of this method. MATERIAL AND METHODS: The frequency of lymph node recurrent disease and complications were prospectively recorded in patients with initially nonpalpable inguinal lymph nodes and histologically negative sentinel lymph nodes. Quality criteria were the false negative rate (percentage of lymph node recurrence in negative procedures) and the morbidity rate. Inguinal regions with palpable lymph nodes and/or evidence of metastases were not considered. RESULTS: The study included 37 patients with histologically negative sentinel lymph nodes in 63 groins with nonpalpable inguinal lymph nodes. There were 21 T1(a/b) stages, 10 T2, and 6 T3 stages. Tumor differentiation was good in 4, moderate in 26, and poor in 7 patients. During a median follow-up of 52 months (range 1-131 months), we observed a bilateral lymph node recurrence in 1 patient and a conservatively managed prolonged lymphorrhea in another patient. Per inguinal region the false-negative rate was 3.2 % and the morbidity rate was 1.6 %; seen per patient the rates were both 2.7 %. CONCLUSIONS: DSNB is a reliable method of lymph node staging in patients with penile carcinoma and nonpalpable inguinal lymph nodes. The high degree of reliability in combination with the low morbidity justifies the higher methodical complexity of this method.

Degradation of conjugated linoleic acid isomers in the yeast Saccharomyces cerevisiae.

Propagation of Saccharomyces cerevisiae cells in conjugated linoleic acid (CLA) medium resulted in activation of the transcriptional machinery that responds to fatty acids. Cells utilized efficiently trans-10,cis-12 CLA, but not the corresponding cis-9,trans-11 isomer, probably due to the formation of cis-3,trans-5-dienoyl-CoA intermediates that are recalcitrant to beta-oxidation.

Saccharomyces cerevisiae Adr1p governs fatty acid beta-oxidation and peroxisome proliferation by regulating POX1 and PEX11.

Saccharomyces cerevisiae Adr1p is essential for fatty acid degradation and peroxisome proliferation. Here, the role of Adr1p was examined with respect to the transcriptional regulation of the Pip2p-Oaf1p dependent genes POX1 and PEX11. POX1 encodes the rate-limiting enzyme of peroxisomal beta-oxidation, acyl-CoA oxidase. The POX1 promoter was shown to contain a canonical Adr1p element (UAS1), within which the oleate response element (ORE) was nested. PEX11 codes for a peroxin that is critical for normal peroxisome proliferation, and its promoter was shown similarly to contain a UAS1-like element overlapping the ORE. Northern analysis demonstrated that transcriptional up-regulation of both POX1 and PEX11 was abolished in adr1 Delta mutant cells, and immunoblotting confirmed that the abundance of their gene products was dramatically reduced. Studies of an overlapping ORE/UAS1 arrangement in the CTA1 promoter revealed synergy between these elements. We conclude that overlapping ORE and UAS1 elements in conjunction with their binding factors Pip2p-Oaf1p and Adr1p coordinate the carbon flux through beta-oxidation with peroxisome proliferation.

The tetratricopeptide repeat domains of human, tobacco, and nematode PEX5 proteins are functionally interchangeable with the analogous native domain for peroxisomal import of PTS1-terminated proteins in yeast.

In the yeast Saccharomyces cerevisiae, beta-oxidation of fatty acids is compartmentalised in peroxisomes. Most yeast peroxisomal matrix proteins contain a type 1C-terminal peroxisomal targeting signal (PTS1) consisting of the tripeptide SKL or a conservative variant thereof. PTS1-terminated proteins are imported by Pex5p, which interacts with the targeting signal via a tetratricopeptide repeat (TPR) domain. Yeast cells devoid of Pex5p are unable to import PTS1-containing proteins and cannot degrade fatty acids. Here, the PEX5-TPR domains from human, tobacco, and nematode were inserted into a TPR-less yeast Pex5p construct to generate Pex5p chimaeras. These hybrid proteins were examined for functional complementation of the pex5delta mutant phenotype. Expression of the Pex5p chimaeras in pex5delta mutant cells restored peroxisomal import of PTS1-terminated proteins. Chimaera expression also re-established degradation of oleic acid, allowing growth on this fatty acid as a sole carbon source. We conclude that, in the context of Pex5p chimaeras, the human, tobacco, and nematode Pex5p-TPR domains are functionally interchangeable with the native domain for the peroxisomal import of yeast proteins terminating with canonical PTS1s. Non-conserved yeast PTS1s, such as HRL and HKL, did not interact with the tobacco PEX5-TPR domain in the two-hybrid system. HRL occurs at the C-terminus of the peroxisomal protein Eci1p, which is required for growth on unsaturated fatty acids. Although mutant pex5delta cells expressing a yeast/tobacco Pex5p chimaera failed to import a GFP-Eci1p reporter protein, they were able to grow on oleic acid. We reason that this is due to a cryptic PTS in native Eci1p that can function in a redundant system with the C-terminal HRL.

Peroxisomal degradation of trans-unsaturated fatty acids in the yeast Saccharomyces cerevisiae.

Degradation of trans-unsaturated fatty acids was studied in the yeast Saccharomyces cerevisiae. Propagation of yeast cells on trans-9 elaidic acid medium resulted in transcriptional up-regulation of the SPS19 gene, whose promoter contains an oleate response element. This up-regulation depended on the Pip2p-Oaf1p transcription factor and was accompanied by induction of import-competent peroxisomes. Utilization of trans fatty acids as a single carbon and energy source was evaluated by monitoring the formation of clear zones around cell growth on turbid media containing fatty acids dispersed with Tween 80. For metabolizing odd-numbered trans double bonds, cells required the beta-oxidation auxiliary enzyme Delta(3)-Delta(2)-enoyl-CoA isomerase Eci1p. Metabolism of the corresponding even-numbered double bonds proceeded in the absence of Sps19p (2,4-dienoyl-CoA reductase) and Dci1p (Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase). trans-2,trans-4-Dienoyl-CoAs could enter beta-oxidation directly via Fox2p (2-enoyl-CoA hydratase 2 and d-specific 3-hydroxyacyl-CoA dehydrogenase) without the involvement of Sps19p, whereas trans-2,cis-4-dienoyl-CoAs could not. This reductase-independent metabolism of trans-2,trans-4-dienoyl-CoAs resembled the situation postulated for mammalian mitochondria in which oleic acid is degraded through a di-isomerase-dependent pathway. In this hypothetical process, trans-2,trans-4-dienoyl-CoA metabolites are generated by Delta(3)-Delta(2)-enoyl-CoA isomerase and Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase and are degraded by 2-enoyl-CoA hydratase 1 in the absence of 2,4-dienoyl-CoA reductase. Growth of a yeast fox2sps19Delta mutant in which Fox2p was exchanged with rat peroxisomal multifunctional enzyme type 1 on trans-9,trans-12 linolelaidic acid medium gave credence to this theory. We propose an amendment to the current scheme of the carbon flux through beta-oxidation taking into account the dispensability of beta-oxidation auxiliary enzymes for metabolizing trans double bonds at even-numbered positions.

Predicting the function and subcellular location of Caenorhabditis elegans proteins similar to Saccharomyces cerevisiae beta-oxidation enzymes.

The role of peroxisomal processes in the maintenance of neurons has not been thoroughly investigated. We propose using Caenorhabditis elegans as a model organism for studying the molecular basis underlying neurodegeneration in certain human peroxisomal disorders, e.g. Zellweger syndrome, since the nematode neural network is well characterized and relatively simple in function. Here we have identified C. elegans PEX-5 (C34C6.6) representing the receptor for peroxisomal targeting signal type 1 (PTS1), defective in patients with such disorders. PEX-5 interacted strongly in a two-hybrid assay with Gal4p-SKL, and a screen using PEX-5 identified interaction partners that were predominantly terminated with PTS1 or its variants. A list of C. elegans proteins with similarities to well-characterized yeast beta-oxidation enzymes was compiled by homology probing. The possible subcellular localization of these orthologues was predicted using an algorithm based on trafficking signals. Examining the C termini of selected nematode proteins for PTS1 function substantiated predictions made regarding the proteins' peroxisomal location. It is concluded that the eukaryotic PEX5-dependent route for importing PTS1-containing proteins into peroxisomes is conserved in nematodes. C. elegans might emerge as an attractive model system for studying the importance of peroxisomes and affiliated processes in neurodegeneration, and also for studying a beta-oxidation process that is potentially compartmentalized in both mitochondria and peroxisomes.

Adr1p-dependent regulation of the oleic acid-inducible yeast gene SPS19 encoding the peroxisomal beta-oxidation auxiliary enzyme 2,4-dienoyl-CoA reductase.

The role of Saccharomyces cerevisiae Adr1p was examined with respect to the transcriptional regulation of the SPS19 gene encoding the peroxisomal beta-oxidation auxiliary enzyme 2,4-dienoyl-CoA reductase. The SPS19 promoter contains both an oleate response element that binds the Pip2p-Oaf1p transcription factor as well as a canonical Adr1p-binding element, termed UAS1(SPS19). Northern analysis demonstrated that transcriptional up-regulation of SPS19 was abolished in cells devoid of Adr1p. Expression of an SPS19-lacZ reporter gene was shown to be quiescent in the adr1Delta mutant and abnormally elevated in cells containing multiple ADR1 copies. UAS1(SPS19) was able to compete for formation of a specific complex between recombinant Adr1p-LacZ and UAS1(CTA1) representing the corresponding Adr1p-binding element in the promoter of the catalase A gene, and to interact directly with this fusion protein. We conclude that in the presence of fatty acids in the medium transcription of SPS19 is directly regulated by both Pip2p-Oaf1p and Adr1p.

A novel element in the promoter of the Saccharomyces cerevisiae gene SPS19 enhances ORE-dependent up-regulation in oleic acid and is essential for de-repression.

In Saccharomyces cerevisiae cells grown on oleic acid, genes encoding enzymes of beta-oxidation are induced by the interaction of a transcription factor composed of Pip2p and Oaflp with an oleate response element (ORE) in their promoters. The SPS19 gene, which encodes peroxisomal 2,4-dienoyl-CoA reductase, an auxiliary beta-oxidation enzyme, has been shown previously to be up-regulated by a canonical ORE. To determine whether additional elements contribute to this transcriptional upregulation, deletion analysis of the SPS19 promoter was conducted using SPS19-lacZ reporter genes. In a reporter construct containing a deletion adjacent to the ORE, transcriptional activation of SPS19 in oleic acid medium was impaired. Together with an additional segment that overlaps a portion of the canonical ORE, this region forms a continuous element (termed UAS(SPS19)) that is essential for de-repression of SPS19 when glucose levels are low. The potentially bi-partite UAS(SPS19) element was able to initiate bi-directional transcription from a promoterless CYC1-lacZ reporter construct under de-repression conditions, whereas the canonical ORE was not. In oleic acid-containing medium, UAS(SPS19) stimulated transcription of the reporter gene 2.4-fold compared to the intact SPS19 ORE, but did so only in the presence of Pip2p and Oaf1p. UAS(SPS19), which is similar to a transcriptional enhancer in the promoter of the sporulation-specific gene SPS4, was shown specifically to bind several proteins, including Pip2p and Oaflp. We propose that UAS(SPS19) and other sequences like it are required to enhance the transcriptional effects mediated by more specific response elements.

Function of human mitochondrial 2,4-dienoyl-CoA reductase and rat monofunctional Delta3-Delta2-enoyl-CoA isomerase in beta-oxidation of unsaturated fatty acids.

Human 2,4-dienoyl-CoA reductase (2,4-reductase; DECR) and rat monofunctional Delta(3)-Delta(2)-enoyl-CoA isomerase (rat 3, 2-isomerase; ECI) are thought to be mitochondrial auxiliary enzymes involved in the beta-oxidation of unsaturated fatty acids. However, their function during this process has not been demonstrated. Although they lack obvious peroxisomal targeting signals (PTSs), both proteins have been suggested previously to also occur in the mammalian peroxisomal compartment. The putative function and peroxisomal location of the two mammalian proteins can be examined in yeast, since beta-oxidation of unsaturated fatty acids is a compartmentalized process in Saccharomyces cerevisiae requiring peroxisomal 2,4-dienoyl-CoA reductase (Sps19p) and peroxisomal 3, 2-isomerase (Eci1p). A yeast sps19Delta mutant expressing human 2, 4-reductase ending with the native C-terminus could not grow on petroselinic acid [cis-C(18:1(6))] medium but could grow when the protein was extended with a PTS tripeptide, SKL (Ser-Lys-Leu). We therefore reason that the human protein is a physiological 2, 4-reductase but that it is probably not peroxisomal. Rat 3, 2-isomerase expressed in a yeast eci1Delta strain was able to re-establish growth on oleic acid [cis-C(18:1(9))] medium irrespective of an SKL extension. Since we had shown that Delta(2,4) double bonds could not be metabolized extra-peroxisomally to restore growth of the sps19Delta strain, we postulate that rat 3,2-isomerase acted on the Delta(3) unsaturated metabolite of oleic acid by replacing the mutant's missing activity from within the peroxisomes. Immunoblotting of fractionated yeast cells expressing rat 3, 2-isomerase in combination with electron microscopy supported our proposal that the protein functioned in peroxisomes. The results presented here shed new light on the function and location of human mitochondrial 2,4-reductase and rat monofunctional 3,2-isomerase.

Alternatives to the isomerase-dependent pathway for the beta-oxidation of oleic acid are dispensable in Saccharomyces cerevisiae. Identification of YOR180c/DCI1 encoding peroxisomal delta(3,5)-delta(2,4)-dienoyl-CoA isomerase.

Fatty acids with double bonds at odd-numbered positions such as oleic acid can enter beta-oxidation via a pathway relying solely on the auxiliary enzyme Delta(3)-Delta(2)-enoyl-CoA isomerase, termed the isomerase-dependent pathway. Two novel alternative pathways have recently been postulated to exist in mammals, and these additionally depend on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (di-isomerase-dependent) or on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase (reductase-dependent). We report the identification of the Saccharomyces cerevisiae oleic acid-inducible DCI1 (YOR180c) gene encoding peroxisomal di-isomerase. Enzyme assays conducted on soluble extracts derived from yeast cells overproducing Dci1p using 3,5,8,11,14-eicosapentenoyl-CoA as substrate demonstrated a specific di-isomerase activity of 6 nmol x min(-1) per mg of protein. Similarly enriched extracts from eci1Delta cells lacking peroxisomal 3,2-isomerase additionally contained an intrinsic 3,2-isomerase activity that could generate 3, 5,8,11,14-eicosapentenoyl-CoA from 2,5,8,11,14-eicosapentenoyl-CoA but not metabolize trans-3-hexenoyl-CoA. Amplification of this intrinsic activity replaced Eci1p since it restored growth of the eci1Delta strain on petroselinic acid for which di-isomerase is not required whereas Eci1p is. Heterologous expression in yeast of rat di-isomerase resulted in a peroxisomal protein that was enzymatically active but did not re-establish growth of the eci1Delta mutant on oleic acid. A strain devoid of Dci1p grew on oleic acid to wild-type levels, whereas one lacking both Eci1p and Dci1p grew as poorly as the eci1Delta mutant. Hence, we reasoned that yeast di-isomerase does not additionally represent a physiological 3,2-isomerase and that Dci1p and the postulated alternative pathways in which it is entrained are dispensable for degrading oleic acid.

The difference in recognition of terminal tripeptides as peroxisomal targeting signal 1 between yeast and human is due to different affinities of their receptor Pex5p to the cognate signal and to residues adjacent to it.

Pex5p is the receptor for the peroxisomal targeting signal 1 (PTS1) that consists of a C-terminal tripeptide (consensus (S/A/C)(K/R/H)(L/M)). Hexadecapeptides recognized by Pex5p from Homo sapiens and Saccharomyces cerevisiae were identified by screening a two-hybrid peptide library, and the targeting ability of the peptides was demonstrated using the green fluorescent protein as reporter. The PTS1 receptors recognized in a species-specific manner a broad range of C-terminal tripeptides, and these are reported herein. In addition, residues upstream of the tripeptide influenced the strength of the interaction in the two-hybrid system as well as in an in vitro competition assay. In peptides interacting with the human protein, hydrophobic residues were found with high frequency especially at positions -2 and -5, whereas peptides interacting with S. cerevisiae Pex5p were more hydrophilic and frequently contained arginine at position -2. In instances where the terminal tripeptide deviated from the consensus, upstream residues exerted a greater influence on the ability of the hexadecapeptides to bind Pex5p.

Peroxisomal Delta3-cis-Delta2-trans-enoyl-CoA isomerase encoded by ECI1 is required for growth of the yeast Saccharomyces cerevisiae on unsaturated fatty acids.

We have identified the Saccharomyces cerevisiae gene ECI1 encoding Delta3-cis-Delta2-trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the beta-oxidation of (poly)unsaturated fatty acids. A mutant devoid of Eci1p was unable to grow on media containing unsaturated fatty acids such as oleic acid but was proficient for growth when a saturated fatty acid such as palmitic acid was the sole carbon source. Levels of ECI1 transcript were elevated in cells grown on oleic acid medium due to the presence in the ECI1 promoter of an oleate response element that bound the transcription factors Pip2p and Oaf1p. Eci1p was heterologously expressed in Escherichia coli and purified to homogeneity. It was found to be a hexameric protein with a subunit of molecular mass 32, 000 Da that converted 3-hexenoyl-CoA to trans-2-hexenoyl-CoA. Eci1p is the only known member of the hydratase/isomerase protein family with isomerase and/or 2-enoyl-CoA hydratase 1 activities that does not contain a conserved glutamate at its active site. Using a green fluorescent protein fusion, Eci1p was shown to be located in peroxisomes of wild-type yeast cells. Rat peroxisomal multifunctional enzyme type I containing Delta3-cis-Delta2-trans-enoyl-CoA isomerase activity was expressed in ECI1-deleted yeast cells, and this restored growth on oleic acid.

Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5.

Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (Nicotiana tabacum) cDNAs encoding Pex5p (NtPEX5) based on the protein's interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.

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

Sporulation in the yeast Saccharormyces cerevisiae is a meiotic developmental process that occurs in MATa/MATalpha 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 delta pex6 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, delta pex6, 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.

Pex14p is a member of the protein linkage map of Pex5p.

To identify members of the translocation machinery for peroxisomal proteins, we made use of the two-hybrid system to establish a protein linkage map centered around Pex5p from Saccharomyces cerevisiae, the receptor for the C-terminal peroxisomal targeting signal (PTS1). Among the five interaction partners identified, Pex14p was found to be induced under conditions allowing peroxisome proliferation. Deletion of the corresponding gene resulted in the inability of yeast cells to grow on oleate as well as the absence of peroxisomal structures. The PEX14 gene product of approximately 38 kDa was biochemically and ultrastructurally demonstrated to be a peroxisomal membrane protein, despite the lack of a membrane-spanning domain. This protein was shown to interact with itself, with Pex13p and with both PTS receptors, Pex5p and Pex7p, indicating a central function for the import of peroxisomal matrix proteins, either as a docking protein or as a releasing factor at the organellar membrane.

The Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductase is encoded by the oleate-inducible gene SPS19.

beta-Oxidation is compartmentalized in mammals into both mitochondria and peroxisomes. Fatty acids with double bonds at even-numbered positions require for their degradation the auxiliary enzyme 2,4-dienoyl-CoA reductase, and at least three isoforms, two mitochondrial and one peroxisomal, exist in the rat. The Saccharomyces cerevisiae Sps19p is 34% similar to the human and rat mitochondrial reductases, and an SPS19 deleted strain was unable to utilize petroselineate (cis-C18:1(6)) as the sole carbon source, but remained viable on oleate (cis-C18:1(9)). Sps19p was purified to homogeneity from oleate-induced cells and the homodimeric enzyme (native molecular weight 69,000) converted 2,4-hexadienoyl-CoA into 3-hexenoyl-CoA in an NADPH-dependent manner and therefore contained 2,4-dienoyl-CoA reductase activity. Antibodies raised against Sps19p decorated the peroxisomal matrix of oleate-induced cells. SPS19 shares with the sporulation-specific SPS18 a common promoter region that contains an oleate response element. This element unidirectionally regulates transcription of the reductase and is sufficient for oleate induction of a promoterless CYC1-lacZ reporter gene. SPS19 is dispensable for growth and sporulation on solid acetate and oleate media, but is essential for these processes to occur on petroselineate.

Constitutive and carbon source-responsive promoter elements are involved in the regulated expression of the Saccharomyces cerevisiae malate synthase gene MLS1.

The malate synthase gene, MLS1, of the yeast Saccharomyces cerevisiae is transcriptionally regulated by the carbon source in the growth medium. A MLS1-lacZ fusion gene, expressed at a basal level in the presence of 2% glucose, is derepressed more than 100-fold under conditions of sugar limitation. No evidence for MLS1 induction by oleic acid was found. By deletion analysis of the MLS1 control region, we identified two sites, UAS1 and UAS2, as important for efficient derepression of the gene. Both sites contain sequences that resemble the previously characterized carbon source-responsive element (CSRE) found in the promoter of the isocitrate lyase gene ICL1. Indeed, UAS1 and UAS2 in the MLS1 upstream region turn out to be functional CSRE sequence variants. This finding allowed us to define a modified version of the CSRE consensus sequence (CCRTYSRNCCG). Protein binding to UAS1MLS1 was observed with extracts from derepressed but not from repressed cells, and could be competed for by an excess of the unlabelled CSRE (ICL1) sequence. No competition was observed with a mutated CSRE variant. Site-directed mutagenesis of both CSREs in the MLS1 promoter reduced gene activation under derepressing conditions to 20% of the wild-type level. The same decrease was observed with the wild-type MLS1 promoter in a cat8 mutant, lacking an activator of CSRE-dependent transcription. The CSRE/Cat8p-independent activation of MLS1 is mediated by constitutive UAS elements. The pleiotropic transcription factor Abf1p, which binds to the MLS1 upstream region, may contribute to constitutive activation. Thus, in order to ensure the severe glucose repression of MLS1 observed, repressor elements that respond to the carbon source must counteract constitutive activation. In summary, ICL1 and MLS1 share common cis-acting elements, although a distinct mechanism of carbon source control also contributes to MLS1 regulation.

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.