Ras is active throughout the cell cycle,but is able to induce cyclin D1 only during G2 phase

The control of cell cycle progression has been studied in asynchronous cultures using image analysis and time lapse techniques. This approach allows determination of the cycle phase and signaling properties of individual cells, and avoids the need for synchronization. In past studies this approach demonstrated that continuous cell cycle progression requires the induction of cyclin D1 levels by Ras, and that this induction takes place during G2 phase. These studies were designed to understand how Ras could induce cyclin D1 levels only during G2 phase. First, in studies with a Ras-specific promoter and cellular migration we find that endogenous Ras is active in all cell cycle phases of actively cycling NIH3T3 cells. This suggests that cyclin D1 induction during G2 phase is not the result of Ras activation specifically during this cell cycle period. To confirm this suggestion oncogenic Ras, which is expected to be active in all cell cycle phases, was microinjected into asynchronous cells. The injected protein induced cyclin D1 levels rapidly, but only in G2 phase cells. We conclude that in the continuously cycling cell the targets of Ras activity are controlled by cell cycle phase, and that this phenomenon is vital to cell cycle progression.     

The relationship between nodulin gene expression and the Rhizobium nod genes in Vicia sativa root nodule development

The role of the Rhizobium nod genes in the induction of nodulin gene expression was examined by analyzing nodules formed on vetch roots by bacterial strains containing only the nod region. Introduction of an 11-kb cloned nod region of the R. leguminosarum sym plasmid pRL1JI into sym plasmid-cured rhizobia conferred on the recipient strains the ability to induce nodules in which all nodulin genes were expressed. This proves that from the sym plasmid only the nod region is involved in the induction of nodulin gene expression. A transconjugant of Agrobacterium carrying the same nod region induces nodules in which only early nodulin gene expression is detected. Thus, the nod region is essential for the induction of early nodulin gene expression. In this case, nodule cytology may indicate that a defense response of the plant interferes with the induction of late nodulin gene expression. Indirect evidence is presented that indeed the Rhizobium nod genes are also in some way involved in the induction of the expression of late noduling genes. The combination between histological data and pattern of nodulin gene expression furthermore reveals a correlation between nodule structure and nodulin gene expression. This correlation may aid in speculations about the functions of nodulins.     

Auxin influx activity is associated with Frankia infection during actinorhizal nodule formation in Casuarina glauca

Plants from the Casuarinaceae family enter symbiosis with the actinomycete Frankia leading to the formation of nitrogen-fixing root nodules. We observed that application of the auxin influx inhibitor 1-naphtoxyacetic acid perturbs actinorhizal nodule formation. This suggests a potential role for auxin influx carriers in the infection process. We therefore isolated and characterized homologs of the auxin influx carrier (AUX1-LAX) genes in Casuarina glauca. Two members of this family were found to share high levels of deduced protein sequence identity with Arabidopsis (Arabidopsis thaliana) AUX-LAX proteins. Complementation of the Arabidopsis aux1 mutant revealed that one of them is functionally equivalent to AUX1 and was named CgAUX1. The spatial and temporal expression pattern of CgAUX1 promoter:beta-glucuronidase reporter was analyzed in Casuarinaceae. We observed that CgAUX1 was expressed in plant cells infected by Frankia throughout the course of actinorhizal nodule formation. Our data suggest that auxin plays an important role during plant cell infection in actinorhizal symbioses.     

CERBERUS, a novel U-box protein containing WD-40 repeats, is required for formation of the infection thread and nodule development in the legume–Rhizobium symbiosis

Endosymbiotic infection of legume plants by Rhizobium bacteria is initiated through infection threads (ITs) which are initiated within and penetrate from root hairs and deliver the endosymbionts into nodule cells. Despite recent progress in understanding the mutual recognition and early symbiotic signaling cascades in host legumes, the molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis are still poorly understood. We isolated a novel symbiotic mutant of Lotus japonicus , cerberus , which shows defects in IT formation and nodule organogenesis. Map-based cloning of the causal gene allowed us to identify the CERBERUS gene, which encodes a novel protein containing a U-box domain and WD-40 repeats. CERBERUS expression was detected in the roots and nodules, and was enhanced after inoculation of Mesorhizobium loti . Strong expression was detected in developing nodule primordia and the infected zone of mature nodules. In cerberus mutants, Rhizobium colonized curled root hair tips, but hardly penetrated into root hair cells. The occasional ITs that were formed inside the root hair cells were mostly arrested within the epidermal cell layer. Nodule organogenesis was aborted prematurely, resulting in the formation of a large number of small bumps which contained no endosymbiotic bacteria. These phenotypic and genetic analyses, together with comparisons with other legume mutants with defects in IT formation, indicate that CERBERUS plays a critical role in the very early steps of IT formation as well as in growth and differentiation of nodules.     

Involvement of the c-Ski oncoprotein in cell cycle arrest and transformation during nurse cell formation after Trichinella spiralis infection

The role of c-Ski, an oncoprotein encoded by the oncogene, c-ski, in Trichinella spiralis-infected muscle tissues during nurse cell formation, was investigated by following the expression kinetics and distribution of c-Ski (both protein and mRNA) in the infected muscle cell, as well as the expression kinetics of the transforming growth factor beta (TGF-beta) signaling pathway factor genes (TGF-beta, Smad2 and Smad4) which cooperate with c-Ski. Immunohistochemical analysis using an anti-c-Ski antibody indicated that in the early stages of infection (13 and 18 days post-infection (p.i.)) the increased expression of the c-Ski protein was limited to the eosinophilic cytoplasm and not the enlarged nuclei or basophilic cytoplasm. At a later stage of infection (23 and 28 days p.i.) the c-Ski protein was limited to the enlarged nuclei in the basophilic cytoplasm, rather than the eosinophilic cytoplasm. At 48 days p.i., the c-Ski protein was barely detectable. Real-time PCR analysis showed that expression of the c-ski gene increased from 13 days p.i., reached a peak at 23-28 days p.i. and then decreased to a low level by 48 days p.i. Expression kinetics for the TGF-beta signaling pathway factor genes (TGF-beta, Smad2 and Smad4) were similar to that of c-ski. These findings provide evidence that the c-Ski protein is involved in nurse cell formation through the TGF-beta signaling pathway process in the host cell nucleus.     

Nitric oxide is required for, and promotes auxin-mediated activation of, cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures

It is now well established that nitric oxide (NO) serves as a signaling molecule in plant cells. In this paper experimental data are presented which indicate that NO can stimulate the activation of cell division and embryogenic cell formation in leaf protoplast-derived cells of alfalfa in the presence of auxin. It was found that various NO-releasing compounds promoted auxin-dependent division (as shown by incorporation of bromodeoxyuridine) of leaf protoplast-derived alfalfa cells. In contrast, application of NO scavenger or NO synthesis inhibitor inhibited the same process. Both the promotion and the inhibition of cell cycle activation correlated with the amount and activity of the cognate alfalfa p34cdc2 protein Medsa;CDKA;1,2. The effect of l-NG-monomethyl-L-arginine (L-NMMA) was transient, and protoplast-derived cells spending more than 3 days in culture become insensitive to the inhibitor as far as cell cycle progression was concerned. L-NMMA had no effect on the cell cycle parameters of cycling suspension-cultured cells, but had a moderate transient inhibitory effect on cells re-entering the cell cycle following phosphate starvation. Cycling cultured cells, however, could respond to NO, as indicated by the sodium nitroprusside (SNP)- and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-dependent accumulation of the ferritin protein. Based on these observations, it is hypothesized that L-NMMA-sensitive generation of NO is involved in the activation, but not the progression of the plant cell division cycle. In addition, SNP promoted and L-NMMA delayed the exogenous auxin [2,4-dichlorophenoxyacetic acid (2,4-D)] concentration-dependent formation of embryogenic cell clusters expressing the MsSERK1 gene; this further supports a link between auxin- and NO-dependent signaling pathways in plant cells.     

Identification of NolR, a negative transacting factor controlling the nod regulon in Rhizobium meliloti.

In Rhizobium meliloti, expression of the nodulation genes (nod and nol genes) is under both positive and negative controls. These genes are activated by the products of the three related nodD genes, in conjunction with signal molecules from the host plants. We showed that negative regulation is mediated by a repressor protein, binding to the overlapping nodD1 and nodA as well as to the nodD2 promoters. The encoding gene, termed nolR, was identified and cloned from strain 41. By subcloning, deletion and Tn5 mutagenesis, a region of 594 base-pairs was found to be necessary and sufficient for repressor production in strains of R. meliloti lacking the repressor or in Escherichia coli. Sequence analysis revealed that nolR encodes a 13,349 Da protein, which is in agreement with the molecular weight of the NolR protein, determined after purification by affinity chromatography, utilizing long synthetic DNA multimers of the 21 base-pair conserved repressor-binding sequence. Our data suggest that the native NolR binds to the operator site in dimeric form. The NolR contains a helix-turn-helix motif, which shows homology to the DNA-binding sequences of numerous prokaryotic regulatory proteins such as the repressor XylR or the activator NodD and other members of the LysR family. Comparison of the putative DNA-binding helix-turn-helix motifs of a large number of regulatory proteins pointed to a number of novel regularities in this sequence. Hybridizations with an internal nolR fragment showed that sequences homologous to the nolR gene are present in all R. meliloti isolates tested, even in those that do not produce the repressor. In another species, such as Rhizobium leguminosarum, where NodD is autoregulated, however, such sequences were not detected.     

Rhizobium infection and nodule development in soybean are affected by exposure of the cotyledons to light

The initiation of Rhizobium infections and the development of nodules on the primary root of soybean Glycine max L. Merr cv Williams seedlings are strongly affected by exposure of the cotyledons/hypocotyls to light. Seedlings in plastic growth pouches were inoculated with R. japonicum in dim light and the position of the root tip of each seedling was marked on the face of the pouch. The pouches were covered and kept in the dark for various times before exposing the upper portions of the plants (cotyledons and hypocotyls) to light. Maximum nodulation occurred if the plants were kept in the dark until 1 day after inoculation. The exposure of plants to light 2 days before inoculation reduced the number of nodules by 50% while the number of nodules was reduced by 70% if the plants were kept in the dark until 7 days after inoculation. Anatomical studies revealed that exposure to light prior to inoculation reduced both the number of infection centers with visible infection threads and the number of infections which developed nodule meristems. Plants kept in the dark for 7 days after inoculation formed a normal number of infection threads above the root tip mark, but very few of these infections developed a nodule meristem. It appears that light stimulates soybean to produce substances which can both inhibit the formation of infection threads and enhance the development of nodules from established infection threads. The effects of light on nodulation appear to be expressed independently of the Rhizobium-induced suppression of nodule formation in younger regions of the root.     

The stringent response is required for amino acid and nitrate utilization, nod factor regulation, nodulation, and nitrogen fixation in Rhizobium etli

A Rhizobium etli Tn5 insertion mutant, LM01, was selected for its inability to use glutamine as the sole carbon and nitrogen source. The Tn5 insertion in LM01 was localized to the rsh gene, which encodes a member of the RelA/SpoT family of proteins. The LM01 mutant was affected in the ability to use amino acids and nitrate as nitrogen sources and was unable to accumulate (p)ppGpp when grown under carbon and nitrogen starvation, as opposed to the wild-type strain, which accumulated (p)ppGpp under these conditions. The R. etli rsh gene was found to restore (p)ppGpp accumulation to a DeltarelA DeltaspoT mutant of Escherichia coli. The R. etli Rsh protein consists of 744 amino acids, and the Tn5 insertion in LM01 results in the synthesis of a truncated protein of 329 amino acids; complementation experiments indicate that this truncated protein is still capable of (p)ppGpp hydrolysis. A second rsh mutant of R. etli, strain AC1, was constructed by inserting an Omega element at the beginning of the rsh gene, resulting in a null allele. Both AC1 and LM01 were affected in Nod factor production, which was constitutive in both strains, and in nodulation; nodules produced by the rsh mutants in Phaseolus vulgaris were smaller than those produced by the wild-type strain and did not fix nitrogen. In addition, electron microscopy revealed that the mutant bacteroids lacked poly-beta-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.     

The Mesorhizobium loti purB gene is involved in infection thread formation and nodule development in Lotus japonicus

The purB and purH mutants of Mesorhizobium loti exhibited purine auxotrophy and nodulation deficiency on Lotus japonicus. In the presence of adenine, only the purH mutant induced nodule formation and the purB mutant produced few infection threads, suggesting that 5-aminoimidazole-4-carboxamide ribonucleotide biosynthesis catalyzed by PurB is required for the establishment of symbiosis.     

Yeast L double-stranded ribonucleic acid is synthesized during the G1 phase but not the S phase of the cell cycle.

The cytoplasm of Saccharomyces cerevisiae contains two major classes of protein-encapsulated double-stranded ribonucleic acids (dsRNA's), L and M. Replication of L and M dsRNA's was examined in cells arrested in the G1 phase by either alpha-factor, a yeast mating pheromone, or the restrictive temperature for a cell cycle mutant (cdc7). [3H]uracil was added during the arrest periods to cells prelabeled with [14C]uracil, and replication was monitored by determining the ratio of 3H/14C for purified dsRNA's. Like mitochondrial deoxyribonucleic acid, both L and M dsRNA's were synthesized in the G1 arrested cells. The replication of L dsRNA was also examined during the S phase, using cells synchronized in two different ways. Cells containing the cdc7 mutation, treated sequentially with alpha-factor and then the restrictive temperature, enter a synchronous S phase when transferred to permissive temperature. When cells entered the S phase, synthesis of L dsRNA ceased, and little or no synthesis was detected throughout the S phase. Synthesis of L dsRNA was also observed in G1 phase cells isolated from asynchronous cultures by velocity centrifugation. Again, synthesis ceased when cells entered the S phase. These results indicate that L dsRNA replication is under cell cycle control. The control differs from that of mitochondrial deoxyribonucleic acid, which replicates in all phases of the cell cycle, and from that of 2-micron DNA, a multiple-copy plasmid whose replication is confined to the S phase.     

Bax-mediated cell death by the Gax homeoprotein requires mitogen activation but is independent of cell cycle activity.

Tissues with the highest rates of proliferation typically exhibit the highest frequencies of apoptosis, but the mechanisms that coordinate these processes are largely unknown. The homeodomain protein Gax is down-regulated when quiescent cells are stimulated to proliferate, and constitutive Gax expression inhibits cell proliferation in a p21(WAF/CIP)-dependent manner. To understand how mitogen-induced proliferation influences the apoptotic process, we investigated the effects of deregulated Gax expression on cell viability. Forced Gax expression induced apoptosis in mitogen-activated cultures, but quiescent cultures were resistant to cell death. Though mitogen activation was required for apoptosis, neither the cdk inhibitor p21(WAF/CIP) nor the tumor suppressor p53 was required for Gax-induced cell death. Arrest in G1 or S phases of the cell cycle with chemical inhibitors also did not affect apoptosis, further suggesting that Gax-mediated cell death is independent of cell cycle activity. Forced Gax expression led to Bcl-2 down-regulation and Bax up-regulation in mitogen-activated, but not quiescent cultures. Mouse embryonic fibroblasts homozygous null for the Bax gene were refractive to Gax-induced apoptosis, demonstrating the functional significance of this regulation. These data suggest that the homeostatic balance between cell growth and death can be controlled by mitogen-dependent pathways that circumvent the cell cycle to alter Bcl-2 family protein expression.     

Binding to the yeast SwI4,6-dependent cell cycle box, CACGAAA, is cell cycle regulated in vivo.

In Saccharomyces cerevisiae commitment to cell division occurs late in the G1 phase of the cell cycle at a point called Start and requires the activity of the Cdc28 protein kinase and its associated G1 cyclins. The Swi4,6-dependent cell cycle box binding factor, SBF, is important for maximal expression of the G1 cyclin and HO endonuclease genes at Start. The cell cycle regulation of these genes is modulated through an upstream regulatory element termed the SCB (SwI4,6-dependent cell cycle box, CACGAAA), which is dependent on both SWI4 and SWI6. Although binding of SWI4 and SWI6 to SCB sequences has been well characterized in vitro, the binding of SBF in vivo has not been examined. We used in vivo dimethyl sulfate footprinting to examine the occupancy of SCB sequences throughout the cell cycle. We found that binding to SCB sequences occurred in the G1 phase of the cell cycle and was greatly reduced in G2. In the absence of either SWI4 or SWI6, SCB sequences were not occupied at any cell cycle stage. These results suggest that the G1-specific expression of SCB-dependent genes is regulated at the level of DNA binding in vivo.     

Measles virus infection of B lymphocytes permits cellular activation but blocks progression through the cell cycle.

Measles virus infection of unstimulated B lymphocytes suppresses both proliferation and differentiation into immunoglobulin-secreting cells. However, mitogenic stimulation of these infected cells results in cell volume enlargement, rapid RNA synthesis, and the expression of cell surface activation antigens 4F2, HLA-DS, and transferrin receptor. The cellular genes c-myc and histone 2B are induced during early G1 and S phase of the cell cycle, respectively, and viral RNA synthesis can be detected during this interval. However, total RNA synthesis is decreased at 48 h after stimulation, and the histone 2B RNA steady-state level at 48 h is fivefold less than that in uninfected cells. This sequence of events defines an arrest in the G1 phase of the cell cycle in measles virus-infected B cells.