The new RGA locus encodes a negative regulator of gibberellin response in Arabidopsis thaliana.

We have identified a new locus involved in gibberellin (GA) signal transduction by screening for suppressors of the Arabidopsis thaliana GA biosynthetic mutant gal-3. The locus is named RGA for repressor of gal-3. Based on the recessive phenotype of the digenic rga/gal-3 mutant, the wild-type gene product of RGA is probably a negative regulator of GA responses. Our screen for suppressors of gal-3 identified 17 mutant alleles of RGA as well as 10 new mutant alleles at the previously identified SPY locus. The digenic (double homozygous) rga/gal-3 mutants are able to partially repress several defects of gal-3 including stem growth, leaf abaxial trichome initiation, flowering time, and apical dominance. The phenotype of the trigenic mutant (triple homozygous) rga/spy/gal-3 shows that rga and spy have additive effects regulating flowering time, abaxial leaf trichome initiation and apical dominance. This trigenic mutant is similar to wild type with respect to each of these developmental events. Because rga/spy/gal-3 is almost insensitive to GA for hypocotyl growth and its bolting stem is taller than the wild-type plant, the combined effects of the rga and spy mutations appear to allow GA-independent stem growth. Our studies indicate that RGA lies on a separate branch of the GA signal transduction pathway from SPY, which leads us to propose a modified model of the GA response pathway.

Vitamin K3 induces cell cycle arrest and cell death by inhibiting Cdc25 phosphatase.

Our early reports have indicated that vitamin K3 (VK3) exerts antitumour activity by inhibiting Cdk1 activity and overexpressing the c-myc gene to induce an apoptotic cell death. In the present study, we investigated the effect of VK3 on Cdc25 phosphatase, a Cdk1 activator and c-Myc-downstream protein. Increased protein level but decreased activity of Cdc25A phosphatase was found in cervical carcinoma SiHa cells treated with VK3 for 1 h and allowed to recover for 8, 24, 30 or 45 h. The binding of VK3 to Cdc25 phosphatase was proven by incubating [methyl-3H]-VK3 with the 27 kDa-catalytic domain of Cdc25A phosphatase at 35 degrees C for 2 h. We found that VK3 inhibited cyclin E expression at late G1 phase and cyclin A at G1/S transition of the aphidicolin-synchronised SiHa cells, but had no effect on Cdk2 and Cdk4. The inhibition of cyclins E and A expression was associated with cell cycle progression delay in the S phase. These results indicate that binding of VK3 to the catalytic domain of Cdc25 phosphatase results in the formation of inactive, hyperphosphorylated Cdk1 that subsequently induces cell cycle arrest, leading to cell death. These findings suggest a possible therapeutic strategy, with VK3 serving as a potential antagonist to tumours expressing high levels of proteins containing cysteine such as oncogenic Cdc25A phosphatase.

Menadione-induced DNA damage in a human tumor cell line.

The nature and extent of menadione (MD)-induced DNA damage were explored using the human breast cancer cell line MCF-7. Concentration-dependent single-strand (ss) and double-strand (ds) DNA breaks were detected in MD-treated MCF-7 cells using the alkaline- and neutral-elution techniques, respectively. The repair of ss and ds DNA breaks was extensive but not complete after a 6-hr incubation in drug-free medium. Evidence was found for the production of DNA interstrand cross-links in MCF-7 cells treated with the bifunctional alkylating agent, mitomycin C, but not for cells treated with MD. Exposure of MCF-7 cells to etoposide (VP-16), mitoxantrone and camptothecin resulted in the detection of significant amounts of protein-linked DNA breaks, whereas none were found in MD-treated cells. These results support the proposition that MD-induced DNA damage is not likely to be mediated via topoisomerases, nor do significant amounts of protein-linked DNA form in MD-treated cells. Thus, MD serves as a good model for examination of the role of the quinone moiety in DNA damage in relation to redox cycling. Future studies directed at elucidation of the biochemical determinants mediating formation of reactive oxygen species effecting the MD-induced DNA damage are necessary and underway.

Menadione (vitamin K3) enhances the mitogenic signal of epidermal growth factor via extracellular signal-regulated kinases.

The effects of vitamin K3 (VK3) on DNA synthesis, cell proliferation and mitogen-activated protein kinase pathway were investigated in G0-arrested NIH 3T3 fibroblasts. VK3 (5 microM) alone stimulates DNA synthesis by 40% and moderately increases the mitogenic effects of EGF, which is preceded by a rapid phosphorylation of the extracellular signal-regulated kinases (ERKs). At 20 microM, VK3 had an antiproliferative effect. VK3 alone (5 and 50 microM) or in concert with EGF increases the activity of ERK2 (by 2.5 and 5 fold, respectively). Our studies demonstrate that the activation of ERKs by VK3 alone, or VK3 plus EGF can promote either stimulatory or inhibitory effects on the mitogenic signal.

Characteristics of silicon nitride after O2 plasma surface treatment for pH-ISFET applications.

Silicon nitride (Si3N4) sensing gate pH-ion-selective field effect transistors (ISFETs) were treated by 2.54-GHz microwave O2 plasma, the results show the ISFET sensitivity has an advantage up to 24% increment after the plasma treatment. Electron spectroscopy for chemical analysis (ESCA) is used to make sure that the plasma treatment is not just a native oxide cleaning procedure. The samples, which were immobilized with glutaraldehyde used as a bifunctional reagent and 3-aminopropyItriethoxysilane used as an adhesion promoter were studied. The binding force between the glucose oxidase and glutaraldehyde immobilized samples, and the element concentrations of nitrogen in 3-aminopropyltriethoxysilane immobilized samples are higher which were treated by plasma.

Serum antioxidant status of civil aircrew.

Objective. To study the serum antioxidant status in civil aircrew members who had more than 4000 h of cumulative flight hours, therefore exposed to a higher dose of cosmic radiation comparing to the dose received by ground residents. Method. Differences in the serum levels of total antioxidant capacity (TAOC), superoxide dismutase (SOD) and malondialdehyde (MDA) were investigated in 230 crew members, 37 local ground residents in Xinjiang, and 37 ground residents in Tianjin. Result. Significantly higher levels of all the three serum indexes were found in civil aircrew members. Serum levels of TAOC and SOD were higher in Xinjiang than in Tianjin ground residents, although no difference was found for MDA. Positive correlations were observed among the three serum indexes in this study. Conclusion. The elevated serum level of MDA in civil aircrew members deserves a proper attention by health care policy makers.

Human FEM1B is required for Rad9 recruitment and CHK1 activation in response to replication stress.

Human checkpoint kinase 1 (CHK1) is an essential kinase required to preserve genome stability, and is activated by DNA replication blockage through the ataxia-telangiectasia-mutated-and-Rad3-related (ATR)/ATRIP-signaling pathway. In this report, we show that a novel CHK1-interacting protein, FEM1B (human homologue of the Caenorhabditis elegans sex determination fem1 protein), identified by a yeast two-hybrid screen, is involved in the activation of CHK1 by replication stress. Depletion of FEM1B by small interfering RNA in cancer cells impairs the activation of CHK1 kinase activity and attenuates the induction of CHK1 Ser345 phosphorylation upon replication interference. It is to be noted that, CHK2 Thr68 phosphorylation is not altered by FEM1B downregulation. By fractionation, we further demonstrated that FEM1B is able to associate with chromatin, and such association facilitates chromatin loading of the Rad9 protein. Consistently, ATR activity is poorly maintained in FEM1B knockdown cells; and FEM1B-ablated cells are as sensitive to replication block as CHK1-depleted cells. Our study has uncovered an adaptor protein FEM1B, which acts as a bridge linking CHK1 and Rad9, thus facilitating checkpoint signaling induced by replication stress.

The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis.

The first committed step in the gibberellin (GA) biosynthetic pathway is the conversion of geranylgeranyl pyrophosphate (GGPP) through copalyl pyrophosphate (CPP) to ent-kaurene catalyzed by ent-kaurene synthetases A and B. The ga1 mutants of Arabidopsis are gibberellin-responsive male-sterile dwarfs. Biochemical studies indicate that biosynthesis of GAs in the ga1 mutants is blocked prior to the synthesis of ent-kaurene. The GA1 locus was cloned previously using the technique of genomic subtraction. Here, we report the isolation of a nearly full-length GA1 cDNA clone from wild-type Arabidopsis. This cDNA clone encodes an active protein and is able to complement the dwarf phenotype in ga1-3 mutants by Agrobacterium-mediated transformation. In Escherichia coli cells that express both the Arabidopsis GA1 gene and the Erwinia uredovora gene encoding GGPP synthase, CPP was accumulated. This result indicates that the GA1 gene encodes the enzyme ent-kaurene synthetase A, which catalyzes the conversion of GGPP to CPP. Subcellular localization of the GA1 protein was studied using 35S-labeled GA1 protein and isolated pea chloroplasts. The results showed that the GA1 protein is imported into and processed in pea chloroplasts in vitro.

fii, a bacterial locus required for filamentous phage infection and its relation to colicin-tolerant tolA and tolB.

We describe mutations in a new bacterial locus, designated fii, which do not allow the filamentous bacteriophage f1 to infect bacteria harboring the F plasmid. Mutations at this locus do not affect the ability of F plasmid-containing bacteria to undergo conjugation or be infected by the F plasmid-specific RNA phage f2. The filamentous phage can still adsorb to the F sex pilus, but the DNA is unable to enter the bacteria. All fii mutants become tolerant to colicins E1, E2, and E3. Strains with amber mutations in fii also are unable to plaque P1, even though they can be infected with this phage. Mutations in fii also prevent infection of bacteria harboring the N plasmid by the filamentous bacteriophage IKe. The fii locus maps adjacent to tolA, mutants of which demonstrate tolerance to high levels of the E and K colicins. The three genes tolA, tolB, and fii are shown to reside on a 4.3-kilobase fragment of the Escherichia coli chromosome. Each gene has been cloned into a chimeric plasmid and shown to complement, in trans, mutations at the corresponding chromosomal locus. Studies in maxicells show that the product of fii appears to be a 24-kilodalton protein which copurifies with the cell envelope. The product of tolA has been identified tentatively as a 51-kilodalton protein. Data from cloning, Tn5 mutagenesis, and P1 transduction studies are consistent with the gene order sucA-fii-tolA-tolB-aroG near 17 min on the E. coli map.

Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli.

Mutations in fii or tolA of the fii-tolA-tolB gene cluster at 17 min on the Escherichia coli map render cells tolerant to high concentrations of the E colicins and do not allow the DNA of infecting single-stranded filamentous bacteriophages to enter the bacterial cytoplasm. The nucleotide sequence of a 1,854-base-pair DNA fragment carrying the fii region was determined. This sequence predicts three open reading frames sequentially coding for proteins of 134, 230, and 142 amino acids, followed by the potential start of the tolA gene. Oligonucleotide mutagenesis of each open reading frame and maxicell analysis demonstrated that all open reading frames are expressed in vivo. Sequence analysis of mutant fii genes identified the 230-amino acid protein as the fii gene product. Chromosomal insertion mutations were constructed in each of the two remaining open reading frames. The phenotype resulting from an insertion of the chloramphenicol gene into the gene coding for the 142-amino acid protein is identical to that of mutations in fii and tolA. This gene is located between fii and tolA, and we propose the designation of tolQRA for this cluster in which tolQ is the former fii gene and tolR is the new open reading frame. The protein products of this gene cluster play an important role in the transport of large molecules such as the E colicins and filamentous phage DNA into the bacterium.

The GA2 locus of Arabidopsis thaliana encodes ent-kaurene synthase of gibberellin biosynthesis.

The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf. Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to ent-kaurene, which is catalyzed by ent-kaurene synthase (KS). Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype. A genomic clone coding for KS, AtKS, was isolated from A. thaliana using CmKS cDNA as a heterologous probe. The corresponding A. thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein. The fusion protein showed enzymatic activity that converted [3H]copalyl diphosphate to [3H]ent-kaurene. The recombinant AtKS protein derived from the ga2-1 mutant is truncated by 14 kD at the C-terminal end and does not contain significant KS activity in vitro. Sequence analysis revealed that a C-2099 to T base substitution, which converts Gln-678 codon to a stop codon, is present in the AtKS cDNA from the ga2-1 mutant. Taken together, our results show that the GA2 locus encodes KS.

The in vitro and in vivo cytotoxicity of menadione (vitamin K3) against rat transplantable hepatoma induced by 3'-methyl-4-dimethyl-aminoazobenzene.

Menadione (vitamin K3, 2-methyl-1,4-naphthoquinone) is a synthetic derivative of napthoquinone. Its ability to inhibit cell growth in a wide variety of and human tumor cell types, and in rat hepatocytes has been recognized. Using a rat transplantable hepatoma model, we have evaluated the cytotoxic activity of menadione in hepatoma cells. Tumor cells in culture were sensitive to menadione treatment. The ID50 of drug is 3.4 microM as shown by a colorimetric MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Tumor-bearing rats were randomized into the treatment (n = 16) and control (n = 15) groups. Rats in the treatment group received intraperitoneal injection of menadione (10 mg/2 ml) once a week for four times; the control group received 2 ml water instead. None of the control rats survived after the 17th day following the start of treatment, while 5 out of the 16 treated rats responded well and survived long-term (greater than 60 days). Medadione was shown to inhibit actively the growth of hepatoma cells in vitro as well as in vivo.

The DELLA motif is essential for gibberellin-induced degradation of RGA.

RGA and GAI are homologous genes that encode putative transcriptional regulators that repress gibberellin (GA) signaling in Arabidopsis. Previously we showed that the green fluorescent protein (GFP)-RGA fusion protein is localized to the nucleus in transgenic Arabidopsis, and expression of this fusion protein rescues the rga null mutation. The GA signal seems to derepress the GA response pathway by degrading the repressor protein RGA. The GA-insensitive, semidominant, semidwarf gai-1 mutant encodes a mutant protein with a 17-amino acid deletion within the DELLA domain of GAI. It was hypothesized that this mutation turns the gai protein into a constitutive repressor of GA signaling. Because the sequences missing in gai-1 are identical between GAI and RGA, we tested whether an identical mutation (rga-Delta 17) in the RGA gene would confer a phenotype similar to gai-1. We demonstrated that expression of rga-Delta 17 or GFP-(rga-Delta 17) under the control of the RGA promoter caused a GA-unresponsive severe dwarf phenotype in transgenic Arabidopsis. Analysis of the mRNA levels of a GA biosynthetic gene, GA4, showed that the feedback control of GA biosynthesis in these transgenic plants was less responsive to GA than that in wild type. Immunoblot and confocal microscopy analyses indicated that rga-Delta17 and GFP-(rga-Delta 17) proteins were resistant to degradation after GA application. Our results illustrate that the DELLA domain in RGA plays a regulatory role in GA-induced degradation of RGA. Deletion of this region stabilizes the rga-Delta 17 mutant protein, and regardless of the endogenous GA status rga-Delta 17 becomes a constitutively active repressor of GA signaling.

Low-frequency infection of F- bacteria by transducing particles of filamentous bacteriophages.

Filamentous particles containing single-stranded plasmid and bacteriophage DNA are able to infect F- Escherichia coli at frequencies of approximately 10(-6). This infection is dependent on an intact particle and requires the products of the tolQ, tolR, and tolA genes of the bacteria. The addition of CaCl2 can increase the frequency about 100-fold, presumably by increasing the concentration of particles at the bacterial surface.

Developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana.

The GA1 gene of Arabidopsis thaliana encodes ent-kaurene synthase A (KSA), which catalyzes the first committed step in the biosynthetic pathway of the plant hormone gibberellin (GA). Its location in the GA biosynthetic pathway has led to speculation that KSA regulation is one of the controlling steps. However, because KSA activity is so low that it is only measurable in Arabidopsis siliques, GA1 promoter-GUS reporter gene fusions and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were used to examine the expression pattern of GA1. The results from this study indicate that GA1 gene expression is highly regulated during growth and development, and it is restricted to specific cell types at the sites of expression. GA1 promoter activity is highest in rapidly growing tissues, e.g. shoot apicas, root tips, developing flowers and seeds. It is also active in the vascular tissue of some non-growing organs, such as expanded leaves, suggesting that these leaves may be a site of GA synthesis for transport to other organs. It was also found that the first one or two introns in the GA1 gene are required for proper expression. Because of the high degree of regulation, GA1 may act as a gatekeeper, controlling the flow of metabolites into the GA biosynthetic pathway, while the levels of specific bioactive GAs are controlled by other downstream steps.

Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9.4 kbp EcoRI-I and -R (polyhedrin gene) region.

The nucleotide sequence of a 9.4-kbp region including the polyhedrin gene of the C6 strain of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome was determined. These data provide a complete description of the EcoRI-I fragment, which is used to produce transfer vectors for inserting foreign genes into the AcMNPV. Ten potential open reading frames (ORFs) were identified in the complete sequence, on either strand of DNA. The largest of these was 1629 nucleotides in length and was located downstream from the polyhedrin coding sequences, but on the opposite strand of DNA. Northern blot hybridization analysis of ORF 8 (1629) identified an RNA of 2000 nucleotides which was produced in infected cells from 12 hr p.i. and remained until at least 48 hr p.i. S1 nuclease mapping and analysis of cDNA clones located the 3' end of the mRNA at a site 16 nucleotides downstream of the polyhedrin coding sequences. The 5' end of the mRNA was mapped using primer extension analysis of polyadenylated RNA. The mRNA start site was positioned within a late/very late consensus transcription initiation motif (ATAAG), 428 nucleotides upstream from the potential ATG translation initiation codon. The biological significance of the putative gene product was assessed by inserting a synthetic oligonucleotide in the carboxyl terminal coding sequences of ORF 8 (1629) to prematurely terminate translation. Recombinant viruses containing this mutation were not isolated, suggesting that the ORF 1629 gene product is essential for virus replication.

Repressing a repressor: gibberellin-induced rapid reduction of the RGA protein in Arabidopsis.

RGA (for repressor of ga1-3) and SPINDLY (SPY) are likely repressors of gibberellin (GA) signaling in Arabidopsis because the recessive rga and spy mutations partially suppressed the phenotype of the GA-deficient mutant ga1-3. We found that neither rga nor spy altered the GA levels in the wild-type or the ga1-3 background. However, expression of the GA biosynthetic gene GA4 was reduced 26% by the rga mutation, suggesting that partial derepression of the GA response pathway by rga resulted in the feedback inhibition of GA4 expression. The green fluorescent protein (GFP)-RGA fusion protein was localized to nuclei in transgenic Arabidopsis. This result supports the predicted function of RGA as a transcriptional regulator based on sequence analysis. Confocal microscopy and immunoblot analyses demonstrated that the levels of both the GFP-RGA fusion protein and endogenous RGA were reduced rapidly by GA treatment. Therefore, the GA signal appears to derepress the GA signaling pathway by degrading the repressor protein RGA. The effect of rga on GA4 gene expression and the effect of GA on RGA protein level allow us to identify part of the mechanism by which GA homeostasis is achieved.