In and out of the plant cell cycle.
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Cell Cycle↗
Biomedical subjects
H Hirt
Publications and source records attributed to H Hirt.
Developmental and cell cycle regulation of alfalfa nucMs1, a plant homolog of the yeast Nsr1 and mammalian nucleolin.
We report here the isolation and characterization of the nucMs1 alfalfa cDNA, whose predicted amino acid sequence structurally resembles the yeast Nsr1 protein and animal nucleolins. These proteins consist of an N-terminal acidic domain, centrally located RNA recognition motifs (RRMs), and a C-terminal glycine- and arginine-rich domain. In comparison with animal nucleolins that contain four RRMs, NucMs1 more closely resembles the yeast Nsr1 protein, which contains only two RRMs. A NucMs1 C-terminal peptide antibody specifically recognized a 95-kD nucleolar protein in alfalfa cells that changed its localization in a cell cycle-dependent manner. The nucMs1 transcript and p95nucMs1 protein levels correlated with cell proliferation, and nucMs1 gene expression was found to be induced in the G1 phase upon mitogenic stimulation of G0-arrested leaf cells. In situ hybridization analysis of different alfalfa organs during various developmental stages showed that nucMs1 gene expression is highest in root meristematic cells, but it is also found in other meristematic cells of the plant body. nucMs1 expression is tightly linked to cell proliferation but does not depend on a particular cell cycle phase. No nucMs1 expression was observed in cells that had exited the cell cycle and were undergoing differentiation or polar growth, indicating that nucMs1 may not be necessary for processes other than cell proliferation.
Animals↗
MMK2, a novel alfalfa MAP kinase, specifically complements the yeast MPK1 function.
Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated in response to a variety of stimuli. Here we report the isolation of an alfalfa cDNA encoding a functional MAP kinase, termed MMK2. The predicted amino acid sequence of MMK2 shares 65% identity with a previously identified alfalfa MAP kinase, termed MMK1. Both alfalfa cDNA clones encode functional kinases when expressed in bacteria, undergoing autophosphorylation and activation to phosphorylate myelin basic protein in vitro. However, only MMK2 was able to phosphorylate a 39 kDa protein from the detergent-resistant cytoskeleton of carrot cells. The distinctiveness of MMK2 was further shown by complementation analysis of three different MAP kinase-dependent yeast pathways; this revealed a highly specific replacement of the yeast MPK1(SLT2) kinase by MMK2, which was found to be dependent on activation by the upstream regulators of the pathway. These results establish the existence of MAP kinases with different characteristics in higher plants, suggesting the possibility that they could mediate different cellular responses.
Amino Acid Sequence↗
Inflorescence-specific expression of AtK-1, a novel Arabidopsis thaliana homologue of shaggy/glycogen synthase kinase-3.
We report here the isolation of the Arabidopsis thaliana gene AtK-1. The predicted protein sequence of AtK-1 shows 70% identity to the Arabidopsis ASK and alfalfa MsK kinases that are homologs of the Drosophila shaggy and rat GSK-3 serine/threonine protein kinases playing an important role in signal transduction processes in animals. Northern analysis of different organs revealed exclusive expression in inflorescences suggesting an involvement of the AtK-1 kinase in reproduction-specific processes.
Amino Acid Sequence↗
Tyrosine phosphatase signalling in a lower plant: cell-cycle and oxidative stress-regulated expression of the Chlamydomonas eugametos VH-PTP13 gene.
The first evidence for tyrosine phosphatase signalling pathways in plants is presented by characterizing a putative protein tyrosine phosphatase gene from the unicellular green alga Chlamydomonas eugametos. This cDNA, referred to as VH-PTP13, contains an open reading frame specifying a protein with a molecular weight of 30.3 kDa, that has significant homology with a distinct group of dual-specificity phosphatases. The highest homology is found with CL-100, a human stress-response gene that regulates MAPkinase activity. The purified VH-PTP13 protein expressed in E. coli had phosphatase activity and inactivated MAPkinases from alfalfa and tobacco. Nondividing C. eugametos gametes did not express the VH-PTP13 gene whereas synchronously dividing vegetative cells only expressed VH-PTP13 in the early G1-phase of the cycle, implying a function there. When vegetative cells were subjected to oxidative stress, expression of the VH-PTP13 gene was strongly induced, analogous to the human CL-100 gene. Its potential role in plant signalling pathways is discussed.
Amino Acid Sequence↗
A cDNA from Medicago sativa encodes a protein homologous to small GTP-binding proteins.
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DNA, Complementary↗
The D-type alfalfa cyclin gene cycMs4 complements G1 cyclin-deficient yeast and is induced in the G1 phase of the cell cycle.
Cyclins are key regulators of the cell cycle in all eukaryotes. In alfalfa, we have previously isolated three B-type cyclins. The closely related cycMs1 and cycMs2 genes are expressed primarily during the G2 and M phases and are most likely mitotic cyclins; expression of the cycMs3 gene is induced in the G0-to-G1 transition, when cells reenter the cell cycle. By complementation of G1 cyclin-deficient yeast cells, a novel alfalfa cyclin, designated cycMs4, was isolated. The predicted amino acid sequence of the cycMs4 gene is most similar to that of the Arabidopsis cyclin delta 3 gene. CycMs4 and cyclin delta 3 belong to the class of D-type cyclins and contain PEST-rich regions and a retinoblastoma binding motif. When comparing expression levels in different organs, cycMs4 transcripts were present predominantly in roots. Whereas expression of the cycMs4 gene was cell cycle-regulated in suspension-cultured cells, transcription in roots was observed to depend also on the positional context of the cell. When differentiated G0-arrested leaf cells were induced to resume cell division by treatment with plant hormones, cycMs4 transcription was induced before the onset of DNA synthesis. Whereas this induction was preceded by that of the cycMs3 gene, cycMs2 expression occurred later and at the same time as mitotic activity. These data suggest that cycMs4 plays a role in the G1-to-S transition and provide a model to investigate the plant cell cycle at the molecular level.
Amino Acid Sequence↗
cycMs3, a novel B-type alfalfa cyclin gene, is induced in the G0-to-G1 transition of the cell cycle.
Cyclins are key regulators of the cell cycle in all eukaryotes. We have previously isolated two B-type cyclin genes, cycMs1 and cycMs2, from alfalfa that are primarily expressed during the G2-to-M phase transition and are most likely mitotic cyclin genes. Here, we report the isolation of a novel alfalfa cyclin gene, termed cycMs3 (for cyclin Medicago sativa), by selecting for mating type alpha-pheromone-induced cell cycle arrest suppression in yeast. The central region of the predicted amino acid sequence of the cycMs3 gene is most similar to the cyclin box of yeast B-type and mammalian A- and B-type cyclins. In situ hybridization showed that cycMs3 mRNA can be detected only in proliferating cells and not in differentiated alfalfa cells. When differentiated G0-arrested cells were induced to reenter the cell cycle in the G1 phase and resume cell division by treatment with plant hormones, cycMs3 transcript levels increased long before the onset of DNA synthesis. In contrast, histone H3-1 mRNA and cycMs2 transcripts were not observed before DNA replication and mitosis, respectively. In addition, cycMs3 mRNA was found in all stages of the cell cycle in synchronously dividing cells, whereas the cycMs2 and histone H3-1 genes showed a G2-to-M phase- or S phase-specific transcription pattern, respectively. These data suggest that the role of cyclin CycMs3 differs from that of CycMs1 and CycMs2. We propose that CycMs3 helps control reentry of quiescent G0-arrested cells into the G1 phase of the cell cycle.
Amino Acid Sequence↗
Evolution of the Enterococcus faecalis sex pheromone system.
In this article we present data that form the basis of a model of the evolution of the unique Enterococcus faecalis sex pheromone system. The data indicate tht sex pheromone plasmid-encoded genes/functions, which are essential for this highly efficient plasmid collection system, are common among all sex pheromone plasmids; pAM373 seems to be only distantly related to all other pheromone plasmids. These genes appear to be restricted to sex pheromone plasmids (but see below for possible exceptions). However, the ability to produce sex pheromone-like peptides is not restricted to E. faecalis. We argue that the sex pheromone system of E. faecalis developed only once--but into at least 19 different variants--to form a system which is highly specific for this bacterium and is related to virulence.
Adhesins, Bacterial↗
The function of the hypusine-containing proteins of yeast and other eukaryotes is well conserved.
The hypusine-containing protein (Hypp) is highly conserved in evolution, from man to archaebacteria, but is not found in eubacteria. Hypp is essential for the viability for yeast cells, where two forms are encoded by the genes HYP1 and HYP2. The hypusine-containing protein Hyp2p, encoded by the HYP2 gene in yeast, is present under both aerobic and anaerobic conditions, whereas Hyp1p synthesis is restricted to anaerobiosis. hyp1 disruption mutants grown under anaerobic conditions reveal no detectable alteration in phenotype relative to wild-type strains. We demonstrate that either Hyp1p or Hyp2p alone is sufficient for normal growth under both metabolic conditions. Moreover, Hypp from various eukaryotic species (slime mold, alfalfa and man) carries the lysine to hypusine modification when expressed in yeast and can substitute functionally for Hyp2p in strains disrupted for HYP2, indicating a highly conserved function of this protein. In contrast, the archaebacterial Hypp expressed in yeast is neither modified by hypusine, nor does it allow growth of cells deficient for yeast Hypp.
Anaerobiosis↗
Isolation and characterization of phosphoprotein phosphatase 1 from alfalfa.
Protein phosphatases are central regulatory components of diverse processes in eukaryotes and are among the most highly conserved proteins known. In this paper, we report the cloning and sequencing of a type 1 protein phosphatase (pp1Ms) cDNA from alfalfa. Southern analysis indicates the presence of a gene family of PP1 proteins in alfalfa. The pp1Ms open reading frame is very similar to one of five predicted Arabidopsis type 1 protein phosphatases, indicating that different subtypes are individually conserved. Expression of the alfalfa pp1Ms in a temperature-sensitive Schizosaccharomyces pombe PP1 mutant, dis2-11, revealed no complementation, suggesting that PP1Ms is not involved in mitotic regulation. In different plant organs, different pp1Ms transcript levels were observed; in contrast, mRNA levels remained constant in all phases of the cell cycle and in logarithmically growing cells. However, when cells entered stationary phase pp1Ms transcript levels decreased considerably.
Amino Acid Sequence↗
The plant transcription factor TGA1 stimulates expression of the CaMV 35S promoter in Saccharomyces cerevisiae.
We have previously shown that two CRE elements situated on a 31 bp region of the cauliflower mosaic virus (CaMV) 35S promoter activate gene expression in the yeast Saccharomyces cerevisiae and are regulated by cAMP. Studies with the yeast transcription factors GCN4, SKO1 and YAP1, which bind CRE-like sequences, showed no influence on expression of the 35S promoter indicating that a yet unknown factor is involved in activation. Band shift experiments with the 31 bp promoter region revealed binding of similar factors in yeast and plant protein extracts. In a previous study this promoter region was shown to confer tissue-specific expression in plants and to interact with the transcription factor TGA1. To test whether expression of TGA1 in yeast also stimulates transcription of the 35S promoter, we co-transformed yeast cells with a cDNA clone of this transcription factor and a 35S promoter/reporter gene construct. Promoter activity studies revealed that TGA1 confers enhanced expression of a reporter gene under the control of the 35S promoter in yeast cells. Yeast cells that were transformed with a 35S promoter construct that containing a mutated TGA1-binding site showed that both TGA1 and the intact binding site are necessary for this activation. These results suggest that stimulation of the 35S promoter by TGA1 is mediated by competition with an endogenous down-regulating yeast factor that is modulated by the nutritional state of the cells.
Base Sequence↗
MAP kinases: universal multi-purpose signaling tools.
MAP (mitogen-activated protein) kinases are serine/threonine protein kinases and mediate intracellular phosphorylation events linking various extracellular signals to different cellular targets. MAP kinase, MAP kinase kinase and MAP kinase kinase kinase are functional protein kinase units that are conserved in several signal transduction pathways in animals and yeasts. Isolation of all three components was also shown in plants and suggests conservation of a protein kinase module in all eukaryotic cells. In plants, MAP kinase modules appear to be involved in ethylene signaling and auxin-induced cell proliferation. Therefore, coupling of different extracellular signals to different physiological responses is mediated by MAP kinase cascades and appears to have evolved from a single prototypical protein kinase module which has been adapted to the specific requirements of different organisms.
Animals↗
The cDNA sequence encoding an annexin from Medicago sativa.
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Annexins↗
Rhizobium nod factors reactivate the cell cycle during infection and nodule primordium formation, but the cycle is only completed in primordium formation.
Rhizobia induce the formation of root nodules on the roots of leguminous plants. In temperate legumes, nodule organogenesis starts with the induction of cell divisions in regions of the root inner cortex opposite protoxylem poles, resulting in the formation of nodule primordia. It has been postulated that the susceptibility of these inner cortical cells to Rhizobium nodulation (Nod) factors is conferred by an arrest at a specific stage of the cell cycle. Concomitantly with the formation of nodule primordia, cytoplasmic rearrangement occurs in the outer cortex. Radially aligned cytoplasmic strands form bridges, and these have been called preinfection threads. It has been proposed that the cytoplasmic bridges are related to phragmosomes. By studying the in situ expression of the cell cycle genes cyc2, H4, and cdc2 in pea and alfalfa root cortical cells after inoculation with Rhizobium or purified Nod factors, we show that the susceptibility of inner cortical cells to Rhizobium is not conferred by an arrest at the G2 phase and that the majority of the dividing cells are arrested at the G0/G1 phase. Furthermore, the outer cortical cells forming a preinfection thread enter the cell cycle although they do not divide.
Amino Acid Sequence↗
Biochemical, immunological and ultrastructural characterization of aggregation substances encoded by Enterococcus faecalis sex-pheromone plasmids.
The sex-pheromone system of Enterococcus faecalis can be viewed as a unique and highly efficient plasmid-collection mechanism. The contact needed for transfer of the conjugative sex-pheromone plasmids is mediated by an adhesin, called aggregation substance, which is encoded by these plasmids. We show here that for 17 of the 18 sex-pheromone plasmids (pAM373 being the exception) described to date, their adhesins are immunologically related to each other. In each case, we observed the presence of an N-terminal fragment of about 78 kDa in addition to the 137-kDa form of mature aggregation substance. The cross-reactions were different for the various plasmids. In the case of pPD1 the 78-kDa fragment reacted only weakly. The aggregation substance encoded by sex-pheromone plasmid pAD1 (Asa1) was characterized in detail. The conditions used for SDS/PAGE had a drastic influence on the migration behavior of mature aggregation substance and differently migrating, interconvertible forms were identified. Preliminary data indicate that Asa1 might be a glycoprotein. Antibodies were isolated which are directed against the N- and C-terminal parts of aggregation substance. They showed about the same reactivity on Western blots; however, only antibodies directed against the N-terminal part of the aggregation substance could inhibit the bacterial cell/cell contact. The reactions of the two antibody preparations with induced cells of E. faecalis was analyzed by transmission electron microscopy. The results indicated that especially the N-terminal part of aggregation substance is exposed on the cell surface of E. faecalis; the C-terminal part seems to be much less exposed.
Antibodies↗
Isolation and characterization of a phosphoprotein phosphatase type 2A gene from alfalfa.
Phosphoprotein phosphatases are central regulatory components of the cell cycle in eukaryotes. We report the cloning and sequencing of an alfalfa phosphoprotein phosphatase type 2A (pp2aMs) cDNA. The predicted protein sequence shows high similarity to PP2A from Brassica napus, rabbit and Drosophila. No changes in pp2aMs mRNA abundance during the cell cycle were found. During growth of a batch cell culture, mRNA levels decreased gradually. In planta, all organs contained pp2a transcripts but maximal mRNA levels were detected in stems. Since Southern analysis indicated the presence of a small pp2a gene family in alfalfa, it appears that different subtypes may have specialized roles in various tissues and developmental situations which await characterization.
Amino Acid Sequence↗
The plant homologue of MAP kinase is expressed in a cell cycle-dependent and organ-specific manner.
In animals, MAP kinase plays a key role in growth factor-stimulated signalling and in mitosis. The isolation of a Medicago sativa cDNA clone MsK7 which shows 52% identity to animal MAP kinases is reported. The deduced protein sequence shows all the important structural features of MAP kinases and also contains the highly conserved Thr-183 and Tyr-185 residues. Northern analysis of synchronized alfalfa cells showed that the MsK7 kinase gene is expressed at low levels in G1 phase but at higher levels in S and G2 phases of the cell cycle. In the plant, only stems and roots were found to contain MAP kinase MsK7 mRNA. Southern and PCR analyses indicated that alfalfa contains at least four highly related MAP kinase genes.
Amino Acid Sequence↗