pBAR-H3.2, a native-optimized binary vector to bypass transgene silencing in alfalfa.

KEY MESSAGE: A novel genetic tool to bypass transgene silencing in alfalfa.

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

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.

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 mouse model for adolescent alcohol abuse: stunted growth and effects in brain.

BACKGROUND: Adolescent alcohol abuse remains a serious public health concern, with nearly a third of high school seniors reporting heavy drinking in the previous month. METHODS: Using the high ethanol-consuming C57BL/6J mouse strain, we examined the effects of ethanol (3.75 g/kg, IP, daily for 45 days) on body weight and brain region mass (cerebral cortex, cerebellum, corpus callosum) during peri-adolescence (postnatal day [P]25 to 70) or adulthood (P180 to 225) of both males and females. RESULTS: In control peri-adolescent animals, body weight gain was greater in males compared with females. In the peri-adolescent exposure group, ethanol significantly reduced body weight gain to a similar extent in both male and female mice (82 and 84% of controls, respectively). In adult animals, body weight gain was much less than that of the peri-adolescent mice, with ethanol having a small but significant effect in males but not females. Between the control peri-adolescent and adult cohorts (measurements taken at P70 and 225, respectively), there were no significant differences in the mass of the cerebral cortex or the cerebellum from either male or female mice, although the rostro-caudal length of the corpus callosum increased slightly but significantly (6.1%) between these time points. CONCLUSIONS: Ethanol treatment significantly reduced the mass of the cerebral cortex in peri-adolescent (-3.1%), but not adult, treated mice. By contrast, ethanol significantly reduced the length of the corpus callosum in adult (-5.4%), but not peri-adolescent, treated mice. Future studies at the histological level may yield additional details concerning ethanol and the peri-adolescent brain.

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.

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.

Kinetic analysis of histone acetylation turnover and Trichostatin A induced hyper- and hypoacetylation in alfalfa.

Dynamic histone acetylation is a characteristic of chromatin transcription. The first estimates for the rate of acetylation turnover of plants are reported, measured in alfalfa cells by pulse, pulse-chase, and steady-state acetylation labeling. Acetylation turnover half-lives of about 0.5 h were observed by all methods used for histones H3, H4, and H2B. This is consistent with the rate at which changes in gene expression occur in plants. Treatment with histone deacetylase inhibitor Trichostatin A (TSA) induced hyperacetylation at a similar rate. Replacement histone variant H3.2, preferentially localized in highly acetylated chromatin, displayed faster acetyl turnover. Histone H2A with a low level of acetylation was not subject to rapid turnover or hyperacetylation. Patterns of acetate labeling revealed fundamental differences between histone H3 versus histones H4 and H2B. In H3, acetylation of all molecules, limited by lysine methylation, had similar rates, independent of the level of lysine acetylation. Acetylation of histones H4 and H2B was seen in only a fraction of all molecules and involved multiacetylation. Acetylation turnover rates increased from mono- to penta- and hexaacetylated forms, respectively. TSA was an effective inhibitor of alfalfa histone deacetylases in vivo and caused a doubling in steady-state acetylation levels by 4-6 h after addition. However, hyperacetylation was transient due to loss of TSA inhibition. TSA-induced overexpression of cellular deacetylase activity produced hypoacetylation by 18 h treatment with enhanced acetate turnover labeling of alfalfa histones. Thus, application of TSA to change gene expression in vivo in plants may have unexpected consequences.

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 MsH3gl were tested. Constructs of the beta-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.