BAS1 and SOB7 act redundantly to modulate Arabidopsis photomorphogenesis via unique brassinosteroid inactivation mechanisms

Active brassinosteroids (BRs), such as brassinolide (BL) and castasterone (CS), are growth-promoting plant hormones. An Arabidopsis cytochrome P450 monooxygenase (CYP734A1, formerly CYP72B1), encoded by the BAS1 gene, inactivates BRs and modulates photomorphogenesis. BAS1 was identified as the overexpressed gene responsible for a dominant, BR-deficient mutant, bas1-D. This mutant was isolated in an activation-tagged screen designed to identify redundant genes that might not be identified in classic loss-of-function screens. Here we report the isolation of a second activation-tagged mutant with a BR-deficient phenotype. The mutant phenotype is caused by the overexpression of SOB7 (CYP72C1), a homolog of BAS1. We generated single and double null-mutants of BAS1 and SOB7 to test the hypothesis that these two genes act redundantly to modulate photomorphogenesis. BAS1 and SOB7 act redundantly with respect to light promotion of cotyledon expansion, repression of hypocotyl elongation and flowering time in addition to other phenotypes not regulated by light. We also provide biochemical evidence to suggest that BAS1 and SOB7 act redundantly to reduce the level of active BRs, but have unique mechanisms. Overexpression of SOB7 results in a dramatic reduction in endogenous CS levels, and although single null-mutants of BAS1 and SOB7 have the same level of CS as the wild type, the double null-mutant has twice the amount. Application of BL to overexpression lines of BAS1 or SOB7 results in enhanced metabolism of BL, though only BAS1 overexpression lines confer enhanced conversion to 26-OHBL, suggesting that SOB7 and BAS1 convert BL and CS into unique products.     

Neurospora strains harboring mitochondrial disease-associated mutations in iron-sulfur subunits of complex I

We subjected the genes encoding the 19.3-, 21.3c-, and 51-kDa iron-sulfur subunits of respiratory chain complex I from Neurospora crassa to site-directed mutagenesis to mimic mutations in human complex I subunits associated with mitochondrial diseases. The V135M substitution was introduced into the 19.3-kDa cDNA, the P88L and R111H substitutions were separately introduced into the 21.3c-kDa cDNA, and the A353V and T435M alterations were separately introduced into the 51-kDa cDNA. The altered cDNAs were expressed in the corresponding null-mutants under the control of a heterologous promoter. With the exception of the A353V polypeptide, all mutated subunits were able to promote assembly of a functional complex I, rescuing the phenotypes of the respective null-mutants. Complex I from these strains displays spectroscopic and enzymatic properties similar to those observed in the wild-type strain. A decrease in total complex I amounts may be the major impact of the mutations, although expression levels of mutant genes from the heterologous promoter were sometimes lower and may also account for complex I levels. We discuss these findings in relation to the involvement of complex I deficiencies in mitochondrial disease.     

NADPH oxidase homologs are required for normal cell differentiation and morphogenesis in Dictyostelium discoideum

Membrane-associated NADPH oxidase complexes catalyse the production of the superoxide anion radical from oxygen and NADPH. In mammalian systems, NADPH oxidases form a family of at least seven isoforms that participate in host defence and signalling pathways. We report here the cloning and the characterisation of slime mould Dictyostelium discoideum homologs of the mammalian heme-containing subunit of flavocytochrome b (gp91(phox)) (NoxA, NoxB and NoxC), of the small subunit of flavocytochrome b (p22(phox)) and of the cytosolic factor p67(phox). Null-mutants of either noxA, noxB, noxC or p22(phox) show aberrant starvation-induced development and are unable to produce spores. The overexpression of NoxA(myc2) in noxA null strain restores spore formation. Remarkably, the gene alg-2B, coding for one of the two penta EF-hand proteins in Dictyostelium, acts as a suppressor in noxA, noxB, and p22(phox) null-mutant strains. Knockout of alg-2B allows noxA, noxB or p22(phox) null-mutants to return to normal development. However, the knockout of gene encoding NoxC, which contains two penta EF-hands, is not rescued by the invalidation of alg-2B. These data are consistent with a hypothesis connecting superoxide and calcium signalling during Dictyostelium development.     

Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase

Monochlorobimane was used as a model xenobiotic for Arabidopsis to directly monitor the compartmentation of glutathione-bimane conjugates in situ and to quantify degradation intermediates in vitro. Vacuolar sequestration of the conjugate was very fast and outcompeted carboxypeptidation to the gamma-glutamylcysteine-bimane intermediate (gamma-EC-B) by phytochelatin synthase (PCS) in the cytosol. Following vacuolar sequestration, degradation proceeded to cysteine-bimane without intermediate. Only co-infiltration of monochlorobimane with Cd2+ and Cu2+ increased gamma-EC-B formation to 4% and 25%, respectively, within 60 min. The role of PCS under simultaneous heavy metal stress was confirmed by investigation of different pcs1 null-mutants. In the absence of elevated heavy metal concentrations glutathione-conjugates are therefore first sequestered to the vacuole and subsequently degraded with the initial breakdown step being rate-limiting.     

Effect of FAK, DLC-1 gene expression on OVCAR-3 proliferation

The study investigates the effect of FAK, DLC-1 on OVCAR-3 proliferation. FAK gene siRNA vector recombinant plasmid was constructed using RNA interference technique. FAK gene-transfected OVCAR-3 cells, OVCAR-3 cells with DLC-1 gene expression, and OVCAR-3 cells with simultaneous expression of DLC-1 and FAK genes were obtained using gene transfection technology. In addition, siRNA control group and blank control were also given. Effect of FAK, DLC-1 gene expression on OVCAR-3 proliferation was examined by FCM and Cell Counting Kit-8 (CCK-8) methods. Results showed that DLC-1 gene high expression and FAK gene silencing, single silencing FAK gene, and single DLC-1 gene high expression in OVCAR-3 cells may decrease S and G2/M phase proportion of the cell cycle. Moreover, DLC-1 gene high expression and FAK gene silencing in OVCAR-3 cells can display the most significant effect. This confirmed that DLC-1 gene high expression and FAK gene silencing may significantly inhibit the OVCAR-3 cells proliferation. CCK-8 analysis showed that silence FAK gene exprssion or/and increasing DLC-1 gene expression may decrease OVCAR-3 growth rate. Moreover, simultaneous silence the exprssion of FAK gene and high expression of DLC-1 gene can display the most significant effect on OVCAR-3 growth. It can be concluded that downregulation of FAK gene expression or/and upregulation of DLC-1 gene expression can all inhibit the OVCAR-3 growth. Moreover, DLC-1 gene expression and FAK gene silencing can display the most marked inhibitory effect on the OVCAR-3 growth.     

The transforming domain alone of the latent membrane protein of Epstein-Barr virus is toxic to cells when expressed at high levels.

A previously unrecognized activity has been associated with the product of the BNLF-1 gene of Epstein-Barr virus. This gene encodes the latent membrane protein of Epstein-Barr virus. When the gene was expressed at high levels, it was toxic to all cell lines tested, which included six human B-lymphoid lines as well as BALB/3T3, 143/EBNA-1, and HEp-2 cells. The BNLF-1 gene was previously shown to induce anchorage-independent and tumorigenic growth in Rat-1 and BALB/3T3 cells. We demonstrate here that only those mutations in the BNLF-1 gene that score positively in the anchorage-independent growth assay were cytotoxic when expressed at high levels. It is therefore possible that the same activities of the latent membrane protein that are necessary to induce anchorage-independent growth of some rodent cell lines also confer toxicity to many cell lines when expressed at high levels.     

Multiple copies of the pyruvate kinase gene affect yeast cell growth

The Saccharomyces cerevisiae pyruvate kinase gene (PYK1) was transformed into yeast using the multicopy vector pJDB207. Growth rates and PYK1 gene expression levels varied considerably amongst the transformants. Yeast transformants expressing the PYK1 gene at high levels formed small colonies compared with those expressing the gene at relatively low levels. Slow-growing transformants 'reverted' at high frequency to more rapid growth, and this correlated with decreases in PYK1 gene copy number and PYK1 mRNA abundance. This apparent selection against PYK1 over-expression was disrupted by the introduction of a stop codon at the 5'-end of the PYK1 coding region, thus confirming that the growth effects were mediated by the PYK1 gene. However, massive overproduction of pyruvate kinase in yeast, using multiple copies of a PGK:PYK gene fusion, had no significant effect upon cell growth. This suggests that the deleterious effect upon the host yeast cell is mediated by abnormally high levels of the wild-type gene or PYK1 mRNA, rather than by increased pyruvate kinase levels.     

Computational architecture of the yeast regulatory network

The topology of regulatory networks contains clues to their overall design principles and evolutionary history. We find that while in- and out-degrees of a given protein in the regulatory network are not correlated with each other, there exists a strong negative correlation between the out-degree of a regulatory protein and in-degrees of its targets. Such correlation positions large regulatory modules on the periphery of the network and makes them rather well separated from each other. We also address the question of relative importance of different classes of proteins quantified by the lethality of null-mutants lacking one of them as well as by the level of their evolutionary conservation. It was found that in the yeast regulatory network highly connected proteins are in fact less important than their low-connected counterparts.     

Ty Element-Induced Temperature-Sensitive Mutations of Saccharomyces Cerevisiae

Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures. The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene. The hit4-1 mutation was located on chromosome XIII. These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response. The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein. Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family. In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus. The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes. The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome. This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein. The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.     

Proper regulation of cyclic AMP-dependent protein kinase is required for growth, conidiation, and appressorium function in the anthracnose fungus Colletotrichum lagenarium

Colletotrichum lagenarium, the casual agent of anthracnose of cucumber, forms specialized infection structures, called appressoria, during infection. To evaluate the role of cAMP signaling in C. lagenarium, we isolated and functionally characterized the regulatory subunit gene of the cAMP-dependent protein kinase (PKA). The RPK1 gene encoding the PKA regulatory subunit was isolated from C. lagenarium by polymerase chain reaction-based screening. rpk1 mutants, generated by gene replacement, exhibited high PKA activity during vegetative growth, whereas the wild-type strain had basal level activity. The rpk1 mutants showed significant reduction in vegetative growth and conidiation. Furthermore, the rpk1 mutants were nonpathogenic on cucumber plants, whereas they formed lesions when inoculated through wounds. A suppressor mutant showing restored growth and conidiation was isolated from a rpk1 mutant culture. The rpkl-suppressor mutant did not show high PKA activity, unlike the parental rpk1 mutant, suggesting that high PKA activity inhibits normal growth and conidiation. The suppressor mutant, however, was nonpathogenic on cucumber and failed to form lesions, even when inoculated through wounds. The rpk1 and suppressor mutants formed melanized appressoria on the host leaf surface but were unable to generate penetration hyphae. These results suggest that proper regulation of the PKA activity by the RPK1-encoded regulatory subunit is required for growth, conidiation, and appressorium function in C. lagenarium.     

The yeast putative transcriptional repressor RGM1 is a proline-rich zinc finger protein

I have cloned a yeast gene, RGM1, which encodes a proline-rich zinc, finger protein. rgm1 mutants do not show any obvious phenotype but overexpression of RGM1 gene greatly impairs cell growth. The proline-rich region of RGM1 attached to a heterologous DNA binding domain is able to repress the expression of the target gene. RGM1 shares similar zinc finger motifs with the mammalian Egr (early growth response) proteins as well as proline-rich sequences with a high serine and threonine content, suggesting that RGM1 and Egr proteins could have functional similarities.     

Inhibition of the growth of human hepatoma cell line both in vitro and in vivo by transducing CKI gene p21~(WAF-1) with GE7 targeting gene delivery system

The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous gene pCEP-p21WAF-1 into human hepatocellular carcinoma cell both in vitro and in vivo. After in vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. The in vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untrans-fected control. The average tumor weight of the experiment group was (0.083 ?0.043) g, while that of the control group was (0.28110.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous gene pCEP-p21WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growt     

Characterization of a Ustilago maydis gene specifically induced during the biotrophic phase: evidence for negative as well as positive regulation

The phytopathogenic basidiomycete Ustilago maydis requires its host plant, maize, for completion of its sexual cycle. To investigate the molecular events during infection, we used differential display to identify plant-induced U. maydis genes. We describe the U. maydis gene mig1 (for "maize-induced gene"), which is not expressed during yeast-like growth of the fungus, is weakly expressed during filamentous growth in axenic culture, but is extensively upregulated during plant infection. mig1 encodes a small, highly charged protein of unknown function which contains a functional N-terminal secretion sequence and is not essential for pathogenic development. Adjacent to mig1 is a second gene (mdu1) related to mig1, which appears to result from a gene duplication. mig1 gene expression during the infection cycle was assessed by fusing the promoter to eGFP. Expression of mig1 was absent in hyphae growing on the leaf surface but was detected after penetration and remained high during subsequent proliferation of the fungus until teliospore formation. Successive deletions as well as certain internal deletions in the mig1 promoter conferred elevated levels of reporter gene expression during growth in axenic culture, indicative of negative regulation. During fungal growth in planta, sequence elements between positions -148 and -519 in the mig1 promoter were specifically required for high levels of induction, illustrating additional positive control. We discuss the potential applications of mig1 for the identification of inducing compounds and the respective regulatory genes.     

Inactivation of YME2/RNA12, which encodes an integral inner mitochondrial membrane protein, causes increased escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae.

Inactivation of the yeast nuclear gene YMe2 causes an increased rate of DNA escape from mitochondria to the nucleus. Mutations in yme2 also show genetic interactions with yme1, a second gene that affects DNA escape from mitochondria to the nucleus. The yme1 cold-sensitive growth phenotype is suppressed by yme2 mutations. In addition, yme1 yme2 double mutants exhibit a synthetic growth defect on ethanol-glycerol medium at 30 degrees C. YME2 was isolated by complementation of the synthetic growth defect of yme1 yme2 strains and was found to be identical with the previously cloned RNA12 gene. The dominant temperature-sensitive mutation RNA12-1 prevents growth of yeast cells at 37 degrees C. YME2 encodes a protein with a predicted molecular weight of 96,681 and is an integral inner mitochondrial membrane protein. The larger carboxyl-terminal domain of the YME2 gene product faces the intermembrane space. Null alleles of yme2 display the same genetic interactions with yme1 and high rate of DNA escape from mitochondria as do the originally isolated yme2 mutant strains. Disruption of yme2 causes a strain-dependent growth defect on nonfermentable carbon sources.     

A mitogen-activated protein kinase (MPKA) is involved in polarized growth in the filamentous fungus, Aspergillus nidulans

An Aspergillus nidulans kinase gene, which encodes a protein kinase with high similarity to mitogen-activated protein kinases involved in cell wall construction and morphogenesis in yeast species, was cloned and sequenced. Targeted deletion of the Aspergillus nidulans kinase gene indicates that this kinase is involved in germination of conidial spores and polarized growth. These defects were largely remedied on complex high osmolarity media, although abnormal swellings of hyphal tips were still observed. Glycerol (1 M) only supported the growth of compact colonies. The Aspergillus nidulans kinase gene is, thus, required for normal polarized growth at several stages of colony formation in the filamentous fungus A. nidulans.     

Inhibition of the growth of human hepatoma cell line both in vitro and in vivo by transducing CKI gene p21WAF-1 with GE7 targeting gene delivery system

The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous gene pCEP-p21WAF-1 into human hepatocellular carcinoma cell both in vitro and in vivo. After in vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. The in vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untransfected control. The average tumor weight of the experiment group was (0.083 ± 0.043) g, while that of the control group was (0.281± 0.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous gene pCEP-p21WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growth with high efficacy both in vivo and in vitro.