Tamas Kapros

Tissue-dependent enhancement of transgene expression by introns of replacement histone H3 genes of Arabidopsis

Intron-bearing replacement histone H3 genes in Arabidopsis and other plants are highly and constitutively expressed. We demonstrate that the introns located within the 5′-untranslated regions (5′-UTR) of the two Arabidopsis replacement H3 genes will abolish the cell cycle dependence of an endogenous histone H4 promoter. We demonstrate that these introns, functionally combined with their endogenous promoters, could produce the high and constitutive expression of the replacement H3 genes observed in planta. They strongly increase gene expression whatever the promoter, from the strong 35S CaMV promoter to complete and resected promoters of cell cycle-dependent and replacement histone genes. Quantitative analysis of the extent of reporter gene enhancement in different parts of developing transgenic plantlets, ranging from 2-fold to 70-fold, supports the notion that trans-acting factors are responsible for this effect. Such factors appear most abundant in roots.

Key components of cell cycle control during auxin-induced cell division

This review article provides a comprehensive summary of the basic molecular and cellular events underlying the induction of the cell division cycle in auxin-treated somatic plant cells. Various pathways of signal transduction chains are discussed as mediators between auxin receptors and alteration of the gene expression pattern. The central role of calcium as a second messenger is analyzed in relation to its interaction with calmodulin and a variety of protein kinases. Experimental data indicate that the control of the cell cycle in higher plants involves several key elements and regulatory mechanisms common to other eukaryotic cells. Recent results show a complex formation between p34cdc2 kinase and cyclin-like proteins. Furthermore, the cell cycle-dependent changes in the p34cdc2 kinase activity which peak at S- and G2/M-phases suggest functional roles for S- and M-forms of the p34cdc2 or related kinases. The homologues of cdc2and cyclin genes have been cloned from different plant species. The expression of plant cdc2 genes is under transcriptional control in auxin-reactivated cells while high constitutive expression of this gene was found in fast cycling cells grown in suspension culture.

Transformation vector based on promoter and intron sequences of a replacement histone H3 gene. A tool for high, constitutive gene expression in plants.

This study explored the possibility of using non-viral, plant-based gene sequences to create strong and constitutive expression vectors. Replacement histone H3 genes are highly and constitutively expressed in all plants. Sequences of the cloned alfalfa histone H3.2 gene MsH3g1 were tested. Constructs of the β-glucuronidase (GUS) reporter gene were produced with H3.2 gene promoter and intron sequences. Their efficiency was compared with that of the commonly used strong 35S cauliflower mosaic virus promoter in transgenic tobacco plants. Combination of the H3.2 promoter and intron produced significantly higher GUS expression than the strong viral 35S promoter. Histochemical GUS analysis revealed a constitutive pattern of expression. Thus, alfalfa replacement H3 gene sequences can be used instead of viral promoters to drive heterologous gene expression in plants, avoiding perceived risks of viral sequences.

Flo11p adhesin required for meiotic differentiation in Saccharomyces cerevisiae minicolonies grown on plastic surfaces.

Saccharomyces cerevisiae grown on plastic surfaces formed organized structures, termed minicolonies, that consisted of a core of round (yeast-like) cells surrounded by chains of filamentous cells (pseudohyphae). Minicolonies had a much higher affinity for plastic than unstructured yeast communities growing on the same surface. Pseudohyphae at the surface of these colonies developed further into chains of asci. These structures suggest that pseudohyphal differentiation and sporulation are sequential processes in minicolonies. Consistent with this idea, minicolonies grown under conditions that stimulated pseudohyphal differentiation contained higher frequencies of asci. Furthermore, a flo11Δ mutant, which fails to form pseudohyphae, yielded normal sporulation in cultures, but was defective for minicolony sporulation. When minicolonies were dispersed in water and cells were then allowed to settle on the plastic surface, these cells sporulated very efficiently. Taken together, our results suggest that sporulation in minicolonies is stimulated by pseudohyphal differentiation because these pseudohyphae are dispersed from the core of the colony.

Phenotypic plasticity within yeast colonies: differential partitioning of cell fates

Across many phyla, a common aspect of multicellularity is the organization of different cell types into spatial patterns. In the budding yeast Saccharomyces cerevisiae, after diploid colonies have completed growth, they differentiate to form alternating layers of sporulating cells and feeder cells. In the current study, we found that as yeast colonies developed, the feeder cell layer was initially separated from the sporulating cell layer. Furthermore, the spatial pattern of sporulation in colonies depended on the colony’s nutrient environment; in two environments in which overall colony sporulation efficiency was very similar, the pattern of feeder and sporulating cells within the colony was very different. As noted previously, under moderately suboptimal conditions for sporulation—low acetate concentration or high temperature—the number of feeder cells increases as does the dependence of sporulation on the feeder-cell transcription factor, Rlm1. Here we report that even under a condition that is completely blocked sporulation, the number of feeder cells still increased. These results suggest broader implications to our recently proposed “Differential Partitioning provides Environmental Buffering” or DPEB hypothesis.

Cell Differentiation and Spatial Organization in Yeast Colonies: Role of Cell-Wall Integrity Pathway

Many microbial communities contain organized patterns of cell types, yet relatively little is known about the mechanism or function of this organization. In colonies of the budding yeast Saccharomyces cerevisiae, sporulation occurs in a highly organized pattern, with a top layer of sporulating cells sharply separated from an underlying layer of nonsporulating cells. A mutant screen identified the Mpk1 and Bck1 kinases of the cell-wall integrity (CWI) pathway as specifically required for sporulation in colonies. The CWI pathway was induced as colonies matured, and a target of this pathway, the Rlm1 transcription factor, was activated specifically in the nonsporulating cell layer, here termed feeder cells. Rlm1 stimulates permeabilization of feeder cells and promotes sporulation in an overlying cell layer through a cell-nonautonomous mechanism. The relative fraction of the colony apportioned to feeder cells depends on nutrient environment, potentially buffering sexual reproduction against suboptimal environments.

A simple method to make better probes from short DNA fragments.

A detailed method is presented tor the creation of head-to-tail multimers of short blunt restriction fragments, ligaled into a plasmid vector in a singletube: reaction. Random priming of the concatemer insert readily yields hybridization probes of high specificity, unattainable from the short monomer fragments.

Identification of a replication-independent replacement histone H3 in the basidiomycete Ustilago maydis

Ustilago maydis is a haploid basidiomycete with single genes for two distinct histone H3 variants. The solitary U1 gene codes for H3.1, predicted to be a replication-independent replacement histone. The U2 gene is paired with histone H4 and produces a putative replication-coupled H3.2 variant. These predictions were evaluated experimentally. U2 was confirmed to be highly expressed in the S phase and had reduced expression in hydroxyurea, and H3.2 protein was not incorporated into transcribed chromatin of stationary phase cells. Constitutive expression of U1 during growth produced ∼25% of H3 as H3.1 protein, more highly acetylated than H3.2. The level of H3.1 increased when cell proliferation slowed, a hallmark of replacement histones. Half of new H3.1 incorporated into highly acetylated chromatin was lost with a half-life of 2.5 h, the fastest rate of replacement H3 turnover reported to date. This response reflects the characteristic incorporation of replacement H3 into transcribed chromatin, subject to continued nucleosome displacement and a loss of H3 as in animals and plants. Although the two H3 variants are functionally distinct, neither appears to be essential for vegetative growth. KO gene disruption transformants of the U1 and U2 loci produced viable cell lines. The structural and functional similarities of the Ustilago replication-coupled and replication-independent H3 variants with those in animals, in plants, and in ciliates are remarkable because these distinct histone H3 pairs of variants arose independently in each of these clades and in basidiomycetes.

Shrinking Daughters: Rlm1-Dependent G1/S Checkpoint Maintains Saccharomyces cerevisiae Daughter Cell Size and Viability

How do cells ensure everything is set for cell division before they start the process? Piccirillo et al. demonstrate that a checkpoint in budding.... The Rlm1 transcription factor is a target of the cell wall integrity pathway. We report that an rlm1Δ mutant grown on a nonfermentable carbon source at low osmolarity forms cell groups in which a mother cell is surrounded by smaller “satellite-daughter” cells. Mother cells in these groups progressed through repeated rounds of cell division with normal rates of bud growth and genetic stability; however, these cells underwent precocious START relative to wild-type mothers. Thus, once activated, Rlm1 delays the transition from G1 to S, a mechanism we term the cell wall/START (CW/START) checkpoint. The rlm1Δ satellite-cell phenotype is suppressed by deletion of either SLT2, which encodes the kinase that activates Rlm1, or SWI4, which is also activated by Slt2; suggesting that Slt2 can have opposing roles in regulating the START transition. Consistent with an Rlm1-dependent CW/START checkpoint, rlm1Δ satellite daughters were unable to grow or divide further even after transfer to rich medium, but UV irradiation in G1 could partially rescue rlm1Δ satellite daughters in the next division. Indeed, after cytokinesis, these satellite daughters shrank rapidly, displayed amorphous actin staining, and became more permeable. As a working hypothesis, we propose that duplication of an “actin-organizing center” in late G1 may be required both to progress through START and to reestablish the actin cytoskeleton in daughter cells.

Flo11p adhesin required for meiotic differentiation in S. cerevisiae minicolonies grown on plastic surfaces

Saccharomyces cerevisiae grown on plastic surfaces formed organized structures, termed minicolonies, that consisted of a core of round (yeast-like) cells surrounded by chains of filamentous cells (pseudohyphae). Minicolonies had a much higher affinity for plastic than unstructured yeast communities growing on the same surface. Pseudohyphae at the surface of these colonies developed further into chains of asci. These structures suggest that pseudohyphal differentiation and sporulation are sequential processes in minicolonies. Consistent with this idea, minicolonies grown under conditions that stimulated pseudohyphal differentiation contained higher frequencies of asci. Furthermore, a flo11Δ mutant, which fails to form pseudohyphae, yielded normal sporulation in cultures, but was defective for minicolony sporulation. When minicolonies were dispersed in water and cells were then allowed to settle on the plastic surface, these cells sporulated very efficiently. Taken together, our results suggest that sporulation in minicolonies is stimulated by pseudohyphal differentiation because these pseudohyphae are dispersed from the core of the colony.