Genetic Interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during Nuclear Fusion in Saccharomyces cerevisiae

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Δ, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Δ and sec72Δ, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein–protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.

Vacuole Fusion Regulated by Protein Phosphatase 2C in Fission Yeast

The gene ptc4+ encodes one of four type 2C protein phosphatases (PP2C) in the fission yeast Schizosaccharomyces pombe. Deletion of ptc4+ is not lethal; however, Δptc4 cells grow slowly in defined minimal medium and undergo premature growth arrest in response to nitrogen starvation. Interestingly, Δptc4 cells are unable to fuse vacuoles in response to hypotonic stress or nutrient starvation. Conversely, Ptc4 overexpression appears to induce vacuole fusion. These findings reveal a hitherto unrecognized function of type 2C protein phosphatases: regulation of vacuole fusion. Ptc4 localizes in vacuole membranes, which suggests that Ptc4 regulates vacuole fusion by dephosphorylation of one or more proteins in the vacuole membrane. Vacuole function is required for the process of autophagy that is induced by nutrient starvation; thus, the vacuole defect of Δptc4 cells might explain why these cells undergo premature growth arrest in response to nitrogen starvation.

A Mutation in a Novel Yeast Proteasomal Gene, RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

A fusion pore phenotype in mast cells of the ruby-eye mouse

Using patch-clamp capacitance and amperometric techniques, we have identified an exocytotic phenotype that affects the function of the fusion pore, the molecular structure that connects the lumen of a secretory vesicle with the extracellular environment during exocytosis. Direct observation of individual exocytotic events in mast cells from the ruby-eye mouse (ru/ru) showed a 3-fold increase in the fraction and duration of transient fusion events with respect to wild-type mice. The fraction of the total fusion events that were transient increased from 0.22 ± 0.02 (wild type) to 0.65 ± 0.02 (ru/ru), and the average duration of these events increased from 418 ± 32 ms (wild type) to 1207 ± 89 ms (ru/ru). We also show that this phenotype can reduce and delay an evoked secretory response by causing the fusion of vesicles that have been previously emptied by repeated cycles of transient fusion. The exocytotic phenotype that we describe here may be a cause of diseases like platelet storage pool deficiency and prolonged bleeding times for which the ruby-eye mouse serves as an animal model. Furthermore, the identification of the gene causing the fusion pore phenotype reported here will illuminate the molecular mechanisms regulating exocytotic fusion.

Distinct leukemia phenotypes in transgenic mice and different corepressor interactions generated by promyelocytic leukemia variant fusion genes PLZF–RARα and NPM–RARα

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation involving RARα and one of four fusion partners: PML, PLZF, NPM, and NuMA genes. To study the leukemogenic potential of the fusion genes in vivo, we generated transgenic mice with PLZF–RARα and NPM–RARα. PLZF–RARα transgenic animals developed chronic myeloid leukemia-like phenotypes at an early stage of life (within 3 months in five of six mice), whereas three NPM–RARα transgenic mice showed a spectrum of phenotypes from typical APL to chronic myeloid leukemia relatively late in life (from 12 to 15 months). In contrast to bone marrow cells from PLZF–RARα transgenic mice, those from NPM–RARα transgenic mice could be induced to differentiate by all-trans-retinoic acid (ATRA). We also studied RARE binding properties and interactions between nuclear corepressor SMRT and various fusion proteins in response to ATRA. Dissociation of SMRT from different receptors was observed at ATRA concentrations of 0.01 μM, 0.1 μM, and 1.0 μM for RARα–RXRα, NPM–RARα, and PML–RARα, respectively, but not observed for PLZF–RARα even in the presence of 10 μM ATRA. We also determined the expression of the tissue factor gene in transgenic mice, which was detected only in bone marrow cells of mice expressing the fusion genes. These data clearly establish the leukemogenic role of PLZF–RARα and NPM–RARα and the importance of fusion receptor/corepressor interactions in the pathogenesis as well as in determining different clinical phenotypes of APL.

Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase

The functional role of the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in epidermal growth factor (EGF)-induced expression of 12(S)-lipoxygenase gene in human epidermoid carcinoma A431 cells was studied. Coimmunoprecipitation experiments indicated that EGF stimulated interaction between c-Jun and Sp1 in a time-dependent manner. Overexpression of Ha-ras and c-Jun also enhanced the amount of c-Jun binding to Sp1. In addition, the c-Jun dominant negative mutant TAM-67 not only inhibited the coimmunoprecipitated c-Jun binding to Sp1 in a dose-dependent manner in cells overexpressing c-Jun but also reduced promoter activity of the 12(S)-lipoxygenase gene induced by c-Jun overexpression. Treatment of cells with EGF increased the interaction between the Sp1 oligonucleotide and nuclear c-Jun/Sp1 in a time-dependent manner. Furthermore, EGF activated the chimeric promoter consisting of 10 tandem GAL4-binding sites, which replaced the three Sp1-binding sites in the 12(S)lipoxygenase promoter only when coexpressed with GAL4-c-Jun () fusion proteins. These results indicate that the direct interaction between c-Jun and Sp1 induced by EGF cooperatively activated expression of the 12(S)-lipoxygenase gene, and that Sp1 may serve at least in part as a carrier bringing c-Jun to the promoter, thus transactivating the transcriptional activity of 12(S)-lipoxygenase gene.

t(11;22)(q23;q11.2) in acute myeloid leukemia of infant twins fuses MLL with hCDCrel, a cell division cycle gene in the genomic region of deletion in DiGeorge and velocardiofacial syndromes

We examined the MLL genomic translocation breakpoint in acute myeloid leukemia of infant twins. Southern blot analysis in both cases showed two identical MLL gene rearrangements indicating chromosomal translocation. The rearrangements were detectable in the second twin before signs of clinical disease and the intensity relative to the normal fragment indicated that the translocation was not constitutional. Fluorescence in situ hybridization with an MLL-specific probe and karyotype analyses suggested t(11;22)(q23;q11.2) disrupting MLL. Known 5′ sequence from MLL but unknown 3′ sequence from chromosome band 22q11.2 formed the breakpoint junction on the der(11) chromosome. We used panhandle variant PCR to clone the translocation breakpoint. By ligating a single-stranded oligonucleotide that was homologous to known 5′ MLL genomic sequence to the 5′ ends of BamHI-digested DNA through a bridging oligonucleotide, we formed the stem–loop template for panhandle variant PCR which yielded products of 3.9 kb. The MLL genomic breakpoint was in intron 7. The sequence of the partner DNA from band 22q11.2 was identical to the hCDCrel (human cell division cycle related) gene that maps to the region commonly deleted in DiGeorge and velocardiofacial syndromes. Both MLL and hCDCrel contained homologous CT, TTTGTG, and GAA sequences within a few base pairs of their respective breakpoints, which may have been important in uniting these two genes by translocation. Reverse transcriptase-PCR amplified an in-frame fusion of MLL exon 7 to hCDCrel exon 3, indicating that an MLL-hCDCrel chimeric mRNA had been transcribed. Panhandle variant PCR is a powerful strategy for cloning translocation breakpoints where the partner gene is undetermined. This application of the method identified a region of chromosome band 22q11.2 involved in both leukemia and a constitutional disorder.

Inactivation of Saccharomyces cerevisiae OGG1 DNA repair gene leads to an increased frequency of mitochondrial mutants

The OGG1 gene encodes a highly conserved DNA glycosylase that repairs oxidized guanines in DNA. We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria. We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild-type. Using green fluorescent protein (GFP) we show that a GFP–Ogg1 fusion protein is transported to mitochondria. However, deletion of the first 11 amino acids from the N-terminus abolishes the transport of the GFP–Ogg1 fusion protein into the mitochondria. This analysis indicates that the N-terminus of Ogg1 contains the mitochondrial localization signal. We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous, as well as induced, oxidative damage. Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 [encoding an isoform of the Ogg1 protein, also known as endonuclease three-like glycosylase I (Ntg1)] leads to suppression of spontaneously arising mutations in the mitochondrial genome when compared with the ogg1 single mutant or the wild-type. Together, these studies provide in vivo evidence for the repair of oxidative lesions in the mitochondrial genome by human and yeast Ogg1 proteins. Our study also identifies Ogg2 as a suppressor of oxidative mutagenesis in mitochondria.

An efficient system for conditional gene expression in embryonic stem cells and in their in vitro and in vivo differentiated derivatives

We have developed a universally applicable system for conditional gene expression in embryonic stem (ES) cells that relies on tamoxifen-dependent Cre recombinase-loxP site-mediated recombination and bicistronic gene-trap expression vectors that allow transgene expression from endogenous cellular promoters. Two vectors were introduced into the genome of recipient ES cells, successively: (i) a bicistronic gene-trap vector encoding the β-galactosidase/neoR fusion protein and the Cre-ERT2 (Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor) and (ii) a bicistronic gene-trap vector encoding the hygroR protein and the human alkaline phosphatase (hAP), the expression of which is prevented by tandemly repeated stop-of-transcription sequences flanked by loxP sites. In selected clones, hAP expression was shown to be regulated accurately by 4′hydroxy-tamoxifen. Strict hormone-dependent expression of hAP was achieved (i) in vitro in undifferentiated ES cells and embryoid bodies, (ii) in vivo in virtually all the tissues of the 10-day-old chimeric fetus (after injection of 4′hydroxy-tamoxifen to foster mothers), and (iii) ex vivo in primary embryonic fibroblasts isolated from chimeric fetuses. Therefore, this approach can be applied to drive conditional expression of virtually any transgene in a large variety of cell types, both in vitro and in vivo.

Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions

Heterocyst differentiation in the filamentous cyanobacterium Anabaena PCC 7120 requires a functional hetR gene. Increased expression of the hetR gene is seen in developing and mature heterocysts in response to fixed nitrogen limitation. We mapped four likely transcriptional start sites for hetR and identified a specific transcript that is positively autoregulated. By using the copper-responsive petE promoter from Anabaena PCC 7120 to drive hetR expression, we show that ectopic expression of hetR increases heterocyst frequency and induces heterocyst differentiation under fully repressing conditions. Coexpression of a reporter gene shows that expression from the petE promoter is smoothly induced depending on the amount of copper supplied. In the heterocyst pattern mutant PatA, where terminally positioned heterocysts are formed almost exclusively, expression of the petE∷hetR fusion does not result in the formation of intercalary heterocysts. These results suggest that although the intracellular concentration of HetR has to be elevated for the differentiation decision, PatA plays a role as well. This role may be in the form of posttranslational modification of HetR, because PatA is a member of the response regulator family of proteins.

Heterogeneous transcription of an indoleacetic acid biosynthetic gene in Erwinia herbicola on plant surfaces

We investigated the spatial pattern of expression of ipdC, a plant inducible gene involved in indoleacetic acid biosynthesis in Erwinia herbicola, among individual cells on plants to gain a better understanding of the role of this phenotype in the epiphytic ecology of bacteria and the factors involved in the regulation of ipdC. Nonpathogenic E. herbicola strain 299R harboring a transcriptional fusion of ipdC to gfp was inoculated onto bean plants, recovered from individual leaves 48 h after inoculation, and subjected to fluorescence in situ hybridization using a 16S rRNA oligonucleotide probe specific to strain 299R. Epifluorescence images captured through a rhodamine filter were used to distinguish the 5carboxytetramethylrhodamine-labeled cells of strain 299R from other leaf microflora. Quantification of the green fluorescence intensity of individual cells by analysis of digital images revealed that about 65% of the 299R cells recovered from bean leaves had higher ipdC expression than in culture. Additionally, 10% of the cells exhibited much higher levels of green fluorescence than the median fluorescence intensity, indicating that they are more heterogeneous with respect to ipdC expression on plants than in culture. Examination of 299R cells in situ on leaf surfaces by confocal laser scanning microscopy after fluorescence in situ hybridization of cells on leaf samples showed that even cells that were in close proximity exhibited dramatically different green fluorescence intensities, and thus, were in a physical or chemical microenvironment that induced differential expression of ipdC.

Involvement of a human gene related to the Drosophila spen gene in the recurrent t(1;22) translocation of acute megakaryocytic leukemia

The recurrent t(1;22)(p13;q13) translocation is exclusively associated with infant acute megakaryoblastic leukemia. We have identified the two genes involved in this translocation. Both genes possess related sequences in the Drosophila genome. The chromosome 22 gene (megakaryocytic acute leukemia, MAL) product is predicted to be involved in chromatin organization, and the chromosome 1 gene (one twenty-two, OTT) product is related to the Drosophila split-end (spen) family of proteins. Drosophila genetic experiments identified spen as involved in connecting the Raf and Hox pathways. Because almost all of the sequences and all of the identified domains of both OTT and MAL proteins are included in the predicted fusion protein, the OTT-MAL fusion could aberrantly modulate chromatin organization, Hox differentiation pathways, or extracellular signaling.

PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea

We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves.

Role of the iroquois3 homeobox gene in organizer formation

In zebrafish, the organizer is thought to consist of two regions, the yolk syncytial layer (YSL) and the shield. The dorsal YSL appears to send signals that affect formation of the shield in the overlying mesendoderm. We show here that a domain of dorsal deep cells located between the YSL and the shield is marked by expression of the iro3 gene. As gastrulation proceeds, the iro3 positive domain involutes and migrates to the animal pole. Iro3 expression is regulated by Nodal and bone morphogenic protein antagonists. Overexpression of iro3 induced ectopic expression of shield-specific genes. This effect was mimicked by an Iro3-Engrailed transcriptional repressor domain fusion, whereas an Iro3-VP16 activator domain fusion behaved as a dominant negative or antimorphic form. These results suggest that Iro3 acts as a transcriptional repressor and further implicate the iro3 gene in regulating organizer formation. We propose that the iro3-expressing dorsal deep cells represent a distinct organizer domain that receives signals from the YSL and in turn sends signals to the forming shield, thereby influencing its expansion and differentiation.

The Caenorhabditis elegans hif-1 gene encodes a bHLH-PAS protein that is required for adaptation to hypoxia

Hypoxia-inducible factor, a heterodimeric transcription complex, regulates cellular and systemic responses to low oxygen levels (hypoxia) during normal mammalian development or tumor progression. Here, we present evidence that a similar complex mediates response to hypoxia in Caenorhabditis elegans. This complex consists of HIF-1 and AHA-1, which are encoded by C. elegans homologs of the hypoxia-inducible factor (HIF) α and β subunits, respectively. hif-1 mutants exhibit no severe defects under standard laboratory conditions, but they are unable to adapt to hypoxia. Although wild-type animals can survive and reproduce in 1% oxygen, the majority of hif-1-defective animals die in these conditions. We show that the expression of an HIF-1:green fluorescent protein fusion protein is induced by hypoxia and is subsequently reduced upon reoxygenation. Both hif-1 and aha-1 are expressed in most cell types, and the gene products can be coimmunoprecipitated. We conclude that the mechanisms of hypoxia signaling are likely conserved among metazoans. Additionally, we find that nuclear localization of AHA-1 is disrupted in an hif-1 mutant. This finding suggests that heterodimerization may be a prerequisite for efficient nuclear translocation of AHA-1.