Identification and sequence analysis of a rap gene from the true slime mold Physarum polycephalum

A member of the ras gene superfamily, belonging to the rap family and designated Pprap1, was isolated from a cDNA library from the true slime mold Physarum polycephalum by plaque hybridization in combination with 5′-RACE. The assembled nucleotide sequence of Pprap1 (1062 bp) has an open reading frame coding for a protein of 188 amino acids of a calculated M_r of 21035. This protein exhibits: (i) a highly conserved GTP binding domain containing a putative effector domain, with the threonine-for-glutamine substitution characteristic of rap proteins, (ii) a hypervariable domain, and (iii) the CAAX motif. Analysis of the C-terminal amino acid sequence of Pprap1 shows that it presumably undergoes geranylgeranylation but is not palmitoylated; however, it contains a lysine-rich domain which might serve as the second membrane localization signal. Pprap1 exhibits significantly high amino acid homology within the GTP binding domain with its homologues: Ddrap1 from Dictyostelium discoideum (92%) and human Rap1A (83%), and relatively low homology (59%) with the Saccharomyces cerevisiae homologue, RSR1. It has also 59% and 61% homology with the P. polycephalum Ppras1 and Ppras2 proteins, respectively. This gene is the third member of the ras gene superfamily identified in P. polycephalum so far.     

Molecular identification of PpHDAC1, the first histone deacetylase fron the slime mold Physarum polycephalum

The dynamic state of post-translational acetylation of eukaryotic histones is maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs and HDACs have been shown to be components of various regulatory protein complexes in the cell. Their enzymatic activities, intracellular localization and substrate specificities are regulated in a complex, cell cycle related manner. In the myxomycete Physarum polycephalum multiple HATs and HDACs can be distinguished in biochemical terms and they exhibit dynamic activity patterns depending on the cell cycle stage. Here we report on the cloning of the first P. polycephalum HDAC (PpHDAC1) related to the S. cerevisiae Rpd3 protein. The expression pattern of PpHDAC1 mRNA was analysed at different time points of the cell cycle and found to be largely constant. Treatment of macroplasmodia with the HDAC inhibitor trichostatin A at several cell cycle stages resulted in a significant delay in entry into mitosis of treated versus untreated plasmodia. No effect of TSA treatment could be observed on PpHDAC1 expression itself.     

Genetic structure of the mating-type locus of Chlamydomonas reinhardtii.

Portions of the cloned mating-type (MT) loci (mt(+) and mt(-)) of Chlamydomonas reinhardtii, defined as the approximately 1-Mb domains of linkage group VI that are under recombinational suppression, were subjected to Northern analysis to elucidate their coding capacity. The four central rearranged segments of the loci were found to contain both housekeeping genes (expressed during several life-cycle stages) and mating-related genes, while the sequences unique to mt(+) or mt(-) carried genes expressed only in the gametic or zygotic phases of the life cycle. One of these genes, Mtd1, is a candidate participant in gametic cell fusion; two others, Mta1 and Ezy2, are candidate participants in the uniparental inheritance of chloroplast DNA. The identified housekeeping genes include Pdk, encoding pyruvate dehydrogenase kinase, and GdcH, encoding glycine decarboxylase complex subunit H. Unusual genetic configurations include three genes whose sequences overlap, one gene that has inserted into the coding region of another, several genes that have been inactivated by rearrangements in the region, and genes that have undergone tandem duplication. This report extends our original conclusion that the MT locus has incurred high levels of mutational change.     

Quantitation of alpha-factor internalization and response during the Saccharomyces cerevisiae cell cycle.

The alpha-factor pheromone binds to specific cell surface receptors on Saccharomyces cerevisiae a cells. The pheromone is then internalized, and cell surface receptors are down-regulated. At the same time, a signal is transmitted that causes changes in gene expression and cell cycle arrest. We show that the ability of cells to internalize alpha-factor is constant throughout the cell cycle, a cells are also able to respond to pheromone throughout the cycle even though there is cell cycle modulation of the expression of two pheromone-inducible genes, FUS1 and STE2. Both of these genes are expressed less efficiently near or just after the START point of the cell cycle in response to alpha-factor. For STE2, the basal level of expression is modulated in the same manner.     

Role for E2F in Control of Both DNA Replication and Mitotic Functions as Revealed from DNA Microarray Analysis

We have used high-density DNA microarrays to provide an analysis of gene regulation during the mammalian cell cycle and the role of E2F in this process. Cell cycle analysis was facilitated by a combined examination of gene control in serum-stimulated fibroblasts and cells synchronized at G(1)/S by hydroxyurea block that were then released to proceed through the cell cycle. The latter approach (G(1)/S synchronization) is critical for rigorously maintaining cell synchrony for unambiguous analysis of gene regulation in later stages of the cell cycle. Analysis of these samples identified seven distinct clusters of genes that exhibit unique patterns of expression. Genes tend to cluster within these groups based on common function and the time during the cell cycle that the activity is required. Placed in this context, the analysis of genes induced by E2F proteins identified genes or expressed sequence tags not previously described as regulated by E2F proteins; surprisingly, many of these encode proteins known to function during mitosis. A comparison of the E2F-induced genes with the patterns of cell growth-regulated gene expression revealed that virtually all of the E2F-induced genes are found in only two of the cell cycle clusters; one group was regulated at G(1)/S, and the second group, which included the mitotic activities, was regulated at G(2). The activation of the G(2) genes suggests a broader role for E2F in the control of both DNA replication and mitotic activities.     

Valproate-induced teratogenesis in Japanese rice fish (Oryzias latipes) embryogenesis

Fertilized eggs of Japanese rice fish (medaka) at three developmental stages (Iwamatsu stages 4-30) were exposed to waterborne valproic acid (VPA) (0-80 mM) in hatching solution for 48 h. The amount of valproate to cause 50% mortality (IC(50)) is found to be developmental stage-specific. The embryos were more sensitive to valproate at early stages of development (Iwamatsu stages 4-10) than in the embryos in late stages (Iwamatsu stages 17-30). Valproate exposed embryos have microcephaly and disrupted cardiovasculature with delayed vessel circulation, thrombus formation, and slow heart rate. The hatching efficiency is also reduced by valproate exposure due to developmental delay. The mRNA analysis of nine genes belong to oxidative stress (catalase, gsr, gst), neurogenesis (iro3, wnt1, shh, otx2, nlgn3b) and cell cycle regulation (ccna2) have been done. It was observed that the genes belong to oxidative stress remained unaltered after valproate exposure. However, some of the genes belong to neurogenesis (wnt1,shh, otx2 and nlgn3b) and cell cycle (ccna2) showed developmental stage-specific alteration after valproate exposure. This study indicates that valproate is able to induce some of the phenotypic features which are analogous to human fetal valproate syndrome (FVS). Modulation of genes expressed in neural tissues indicates that this fish can be used to analyze the mechanisms of many neurobehavioral disorders like Autism spectrum disorder (ASD) in human.     

Microarray analysis of selected genes in neural stem and progenitor cells

To access and compare gene expression in fetal neuroepithelial cells (NEPs) and progenitor cells, we have used microarrays containing approximately 500 known genes related to cell cycle regulation, apoptosis, growth and differentiation. We have identified 152 genes that are expressed in NEPs and 209 genes expressed by progenitor cells. The majority of genes (141) detected in NEPs are also present in progenitor populations. There are 68 genes specifically expressed in progenitors with little or no expression in NEPs, and a few genes that appear to be present exclusively in NEPs. Using cell sorting, RT-PCR, in situ hybridization or immunocytochemistry, we have examined the segregation of expression to neuronal and glial progenitors, and identified several that appeared to be enriched in neuronal (e.g. CDK5, neuropilin, EphrinB2, FGF11) or glial (e.g. CXCR4, RhoC, CD44, tenascin C) precursors. Our data provide a first report of gene expression profiles of neural stem and progenitor cells at early stages of development, and provide evidence for the potential roles of specific cell cycle regulators, chemokines, cytokines and extracellular matrix molecules in neural development and lineage segregation.     

The effect of cycloheximide on the expression of cell cycle dependent genes

We have investigated the inducibility of several cell cycle-dependent genes (plus control sequences, not expressed in a cell cycle-dependent manner) in the presence of cycloheximide, an inhibitor of protein synthesis. The genes studied include: 1) five cDNA clones that are preferentially expressed in the G1 phase of the cell cycle: KC-1, JE-3, 2F1, 4F1 and 2A9; 2) one gene preferentially expressed in late G1/S phase: histone H3; and 3) the cell cycle-dependent oncogene p53. All the genes studied are induced by serum even in the presence of cycloheximide. Previous results in the literature have shown that 2 other oncogenes, c-myc and c-fos, can be induced by growth factors in the presence of cycloheximide. Together with our results, these findings indicate that protein synthesis is not required for the induction of at least nine cell cycle genes by growth factors.     

Cell cycle dependent genes inducible by different mitogens in cells from different species

Summary A number of genes and cDNA sequences (including at least four oncogenes) are known to be expressed in a cell cycle-dependent manner, i.e. the levels of specific mRNAs vary with the phases of the cell cycle. In order to explore the significance of some of these sequences in the mitogenic response, we have investigated the expression of 8 cell cycle-dependent sequences (plus two control sequences, not expressed in a cell cycle-dependent manner) under a variety of conditions. These conditions included cells of different types, from different species, stimulated to proliferate by different mitogens. The genes (or sequences) studied included five cDNA clones whose sequences are preferentially expressed in early G₁, i.e. two cDNA clones inducible by platelet-derived growth factor (JE-3 and KC-1), and three cDNA clones inducible by serum (2A9, 2F1, 4F1); and three oncogenes (c-myc, c-ras^Ha and p53) whose expression is known to be cell cycle-dependent. All of the tested genes, except 2A9, c-ras^Ha and the control genes, are expressed in a cell cycle-dependent manner in human peripheral blood mononuclear cells stimulated by phytohemagglutinin and in serum-stimulated mouse and Syrian hamster fibroblasts. The inducibility of these genes by different mitogens in cells of different types and from different species strongly suggests that these genes play a role in cell cycle progression. This conclusion is further supported by the known structural and functional similarities between cell-cycle dependent genes, oncogenes and genes coding for cell-cycle related molecules.     

The helix-loop-helix inhibitor of differentiation (ID) proteins induce post-mitotic terminally differentiated Sertoli cells to re-enter the cell cycle and proliferate

Prior to puberty the Sertoli cells undergo active cell proliferation, and at the onset of puberty they become a terminally differentiated postmitotic cell population that support spermatogenesis. The molecular mechanisms involved in the postmitotic block of pubertal and adult Sertoli cells are unknown. The four known helix-loop-helix ID proteins (i.e., Id1, Id2, Id3, and Id4) are considered dominant negative regulators of cellular differentiation pathways and act as positive regulators of cellular proliferation. ID proteins are expressed at low levels by postpubertal Sertoli cells and are transiently induced by serum. The hypothesis tested was that ID proteins can induce a terminally differentiated postmitotic Sertoli cell to reenter the cell cycle if they are constitutively expressed. To test this hypothesis, ID1 and ID2 were stably integrated and individually overexpressed in postmitotic rat Sertoli cells. Overexpression of ID1 or ID2 allowed postmitotic Sertoli cells to reenter the cell cycle and undergo mitosis. The cells continued to proliferate even after 300 cell doublings. The functional markers of Sertoli cell differentiation such as transferrin, inhibin alpha, Sert1, and androgen binding protein (ABP) continued to be expressed by the proliferating Sertoli cells, but at lower levels. FSH receptor expression was lost in the proliferating Sertoli cell-Id lines. Some Sertoli cell genes, such as cyclic protein 2 (cathepsin L) and Sry-related HMG box protein-11 (Sox11) increase in expression. At no stage of proliferation did the cells exhibit senescence. The expression profile as determined with a microarray protocol of the Sertoli cell-Id lines suggested an overall increase in cell cycle genes and a decrease in growth inhibitory genes. These results demonstrate that overexpression of ID1 and ID2 genes in a postmitotic, terminally differentiated cell type have the capacity to induce reentry into the cell cycle. The observations are discussed in regards to potential future applications in model systems of terminally differentiated cell types such as neurons or myocytes.