The FUSCA genes of Arabidopsis: negative regulators of light responses

More than 200 fusca mutants of Arabidopsis have been isolated and characterised, defining 14 complementation groups. Mutations in at least nine FUSCA genes cause light-dependent phenotypic changes in the absence of light: high levels of anthocyanin accumulation in both the embryo and the seedling, inhibition of hypocotyl elongation, apical hook opening, and unfolding of cotyledons. In double mutants, the fusca phenotype is epistatic to the hy phytochromedeficiency phenotype, indicating that the FUSCA genes act downstream of phytochrome. By contrast, the accumulation of anthocyanin is suppressed by mutations in TT and TTG genes, which affect the biosynthesis of anthocyanin, placing the FUSCA genes upstream of those genes. Regardless of the presence or absence of anthocyanin, fusca mutations limit cell expansion and cause seedling lethality. In somatic sectors, mutant fus1 cells are viable; expressing tissue-specific phenotypes: reduced cell expansion and accumulation of anthocyanin in subepidermal tissue, formation of ectopic trichomes but no reduced cell expansion in epidermal tissue. Our results suggest a model of FUSCA gene action in light-induced signal transduction.     

生物钟PRR蛋白促进拟南芥幼苗中花青素的合成

生物钟(circadian clock)是激发植物生理特征节律性表达，并使之维持稳定的保守内源调节机制。PRR(PSEUDO-RESPONSE REGULATOR)蛋白家族是生物钟中央振荡器的重要组成部分，调控植物的种子萌发、下胚轴伸长和开花等多种生命过程。花青素(anthocyanin)是植物次生代谢产物，对植物的繁衍、生长发育和抵抗逆境胁迫具有重要作用。该研究以拟南芥(Arabidopsis thaliana)为对象，探讨生物钟PRR蛋白对花青素生物合成的调控功能和分子机制。结果表明：(1)在PRR基因单突变体及多突变体幼苗中，花青素的积累明显降低，某些花青素合成相关基因的表达也显著降低。(2)相反，在PRR5过表达幼苗中，花青素的积累以及某些花青素合成相关基因的表达则显著升高。(3)蛋白相互作用结果显示，PRR5蛋白能与MYB75、TT8、MYB90及MYB113等花青素调控蛋白相互作用，并形成复合物。(4)遗传学分析结果显示，拟南芥PRR5诱导幼苗中花青素的合成依赖于MYB家族花青素调控蛋白。综上认为，生物钟PRR蛋白可能通过PRR5与MYB75、TT8等相互作用，促进拟南芥幼...     

Ac/Ds transposon mutagenesis in Arabidopsis thaliana: mutant spectrum and frequency of Ds insertion mutants

Using a two-component Ac/Ds system consisting of a stabilized Ac element (Ac_c1) and a non-autonomous element (Ds_A), 650 families of plants carrying independent germinal Ds_A excisions/transpositions were isolated. Progenies of 559 of these Ac_c1/Ds_A families, together with 43 families of plants selected for excision/transposition of wild-type (wt)Ac, were subjected to a broad screening program for mutants exhibiting visible alterations. This resulted in the identification of 48 mutants showing a wide variety of mutant phenotypes, including embryo lethality (24 mutants), chlorophyll defects (5 mutants), defective seedlings (2 mutants), reduced fertility (5 mutants), reduced size (3 mutants), altered leaf morphology (2 mutants), dark green, unexpanded rosette leaves (3 mutants), and aberrant flower or shoot morphology (4 mutants). To test whether these mutants were due to transposon insertions, a series of Southern blot experiments was performed on 28 families, comparing in each case several mutant plants with others showing the wild-type phenotype. A preliminary analysis revealed in 4 of the 28 families analyzed a common, novel Ds_A fragment in all mutant plants, which was present only in heterozygous plants with wt phenotype, as expected for Ds_A insertion mutations. These four mutants included two showing embryo lethality, one with dark green, unexpanded rosette leaves and stunted inflorescences, and one with curly growth of stems, leaves and siliques. Further evidence for Ds_A insertion mutations was obtained for one embryo lethal mutant and for the stunted mutant, while in case of the second embryo lethal mutant, the Ds_A insertion could be separated from the mutant locus by genetic recombination.     

Mutations in somePolycomb group genes ofDrosophila interfere with regulation of segmentation genes

Mutations in severalPolycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting thatPc group genes may regulate the segmentation genes ofDrosophila. We show that individuals doubly heterozygous for mutations inpolyhomeotic and six otherPc group genes show gap, pair rule, and segment polarity segmentation defects. We examined double heterozygous combinations ofPc group and segmentation mutations for enhancement of adult and embryonic segmentation defects.Posterior sex combs andpolyhomeotic interact withKrüppel ² and enhance embryonic phenotypes ofhunchback andknirps, andpolyhomeotic enhanceseven-skipped. Surprisingly, flies carrying duplications ofextra sex combs (esc), that were heterozygous for mutations ofeven-skipped (eve), were extremely subvital. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments. Antibody studies confirm that expression ofeve is suppressed by duplications ofesc. However,esc duplications have no effect on other gap or pair rule genes tested. To our knowledge, this is only the second triplo-abnormal phenotype associated withPc group genes. Duplications of nine otherPc group genes have no detectable effect oneve. Expression ofengrailed (en) was abnormal in the central nervous systems of mostPc group mutants. These results support a role forPc genes in regulation of some segmentation genes, and suggest thatesc may act differently from otherPc group genes.     

Nuclear genes required for post-translational steps in the biogenesis of the chloroplast cytochrome b6f complex in maize

Nuclear genes essential for the biogenesis of the chloroplast cytochrome b ₆ f complex were identified by mutations that cause the specific loss of the complex. We describe four transposon-induced maize mutants that lack cytochrome b ₆ f proteins but contain normal levels of other photosynthetic complexes. The four mutations define two nuclear genes. To identify the step at which each mutation blocks protein accumulation, mRNAs encoding each subunit were examined by Northern hybridization analysis and the rates of subunit synthesis were examined in pulse-labeling experiments. In each mutant the mRNAs encoding the known subunits of the complex were normal in size and abundance and the major subunits were synthesized at normal rates. Thus, these mutations block the biogenesis of the cytochrome b ₆ f complex at a post-translational step. The two nuclear genes identified by these mutations may encode previously unknown subunits, be involved in prosthetic group synthesis or attachment, or facilitate assembly of the complex. These mutations were also used to provide evidence for the authenticity of a proposed fifth subunit of the complex and to demonstrate a role for the cytochrome b ₆ f complex in protecting photosystem 11 from light-induced degradation.     

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.     

茄科作物青枯菌NADH脱氢酶F亚基在细胞运动和致病性中的功能研究

[目的]劳尔氏菌（Ralstonia solanacearum）在茄科作物上引起严重的细菌性青枯病,本研究旨在发掘青枯劳尔氏菌与致病相关的基因。[方法]利用Tn5转座子构建随机插入突变体,分析生物膜形成、细胞运动和致病性;对有表型变化的突变体,运用TAIL-PCR方法鉴定Tn5插入位点,确定所突变的基因。[结果]以模式菌株GMI000为出发菌,总共获得了400个突变体,其中2个突变体不能形成生物膜,在软琼脂平板上的运动能力下降;接种感病番茄植物,这2个突变体都不能引起萎焉症状。TAIL-PCR结果显示,2个突变体的Tn5插入位点都在NADH脱氢酶F亚基（nuoF）中,距离翻译起始位点分别为103-bp和225-bp。ripAY基因启动子推动的nuoF基因互补载体,完全恢复了2个突变体的表型。[结论]NADH脱氢酶复合物是微生物呼吸电子传递链中的第一步催化酶。我们的结果表明,NADH脱氢酶复合物对R.solanacearum生物膜形成、细胞运动和致病性也有重要作用。     

Genetic interactions and dosage effects of Polycomb group genes of Drosophila

The Polycomb (Pc) group of genes are required for maintenance of cell determination in Drosophila melanogaster. At least 11 Pc group genes have been described and there may be up to 40; all are required for normal regulation of homeotic genes, but as a group, their phenotypes are rather diverse. It has been suggested that the products of Pc group genes might be members of a heteromeric complex that acts to regulate the chromatin structure of target loci. We examined the phenotypes of adult flies heterozygous for every pairwise combination of Pc group genes in an attempt to subdivide the Pc group functionally. The results support the idea that Additional sex combs (Asx), Pc, Polycomblike (Pcl), Posterior sex combs (Psc), Sex combs on midleg (Scm), and Sex combs extra (Sce) have similar functions in some imaginal tissues. We show genetic interactions among extra sex combs (esc) and Asx, Enhancer of Pc, Pcl, Enhancer of zeste E(z), and super sex combs and reassess the idea that most Pc group genes function independently of esc. Most duplications of Pc group genes neither exhibit anterior transformations nor suppress the extra sex comb phenotype of Pc group mutations, suggesting that not all Pc group genes behave as predicted by the mass-action model. Surprisingly, duplications of E(z) enhance homeotic phenotypes of esc mutants. Flies with increasing doses of esc ⁺ exhibit anterior transformations, but these are not enhanced by mutations in trithorax group genes. The results are discussed with respect to current models of Pc group function.     

Inactivation of SSM4, a new Saccharomyces cerevisiae gene, suppresses mRNA instability due to rna14 mutations

Decay rates of mRNAs depend on many elements and among these, the role of the poly(A) tail is now well established. In the yeast Saccharomyces cerevisiae, thermosensitive mutations in two genes, RNA14 and RNA15, result in mRNAs having shorter poly(A) tails and reduced half-life. To identify other components interacting in the same process, we have used a genetic approach to isolate mutations that suppress the thermosensitivity of an rna14 mutant strain. Mutations in a single locus, named SSM4, not only suppress the cell growth phenotype but also the mRNA instability and extend the short mRNA poly(A) tails. The frequency of appearance and the recessive nature of these mutations suggested that the suppressor effect was probably due to a loss of function. We failed to clone the SSM4 gene directly by complementation, owing to its absence from gene banks; it later emerged that the gene is toxic to Escherichia coli, but we have nevertheless been able to clone the SSM4 sequence by Ty element transposition tagging. Disruption of the SSM4 gene does not affect cell viability and suppresses the rna14 mutant phenotypes. The protein encoded by the SSM4 gene has a calculated molecular mass of 151 kDa and does not contain any known motif or show homology with known proteins. The toxicity of the SSM4 gene in E. coli suggests that a direct biochemical activity is associated with the corresponding protein.     

Characterization of a region of the X chromosome of Drosophila including multi sex combs (mxc), a Polycomb group gene which also functions as a tumour suppressor

Genetic analysis of the 8D3;8D8-9 segment of the Drosophila melanogaster X chromosome has assigned seven complementation groups to this region, three of which are new. A Polycomb group (Pc-G) gene, multi sex combs (mxc), is characterized and mutant alleles are described. Besides common homeotic transformations characteristic of Pc-G mutants that mimic the ectopic gain of function of BX-C and ANT-C genes, mxc mutants show other phenotypes: they zygotically mimic, in males and females, the characteristic lack of germ line seen in progeny of some maternal effect mutants of the so-called posterior group (the grandchildless phenotype). Loss of normal mxc function can promote uncontrolled malignant growth which indicates a possible relationship between Pc-G genes and tumour suppressor genes. We propose that gain-of-function of genes normally repressed by the wild-type mxc product could, in mxc mutants, give rise to an incoherent signal which would be devoid of meaning in normal development. Such a signal could divert somatic and germ line developmental pathways, provoke the loss of cell affinities, but allow or promote growth.     

Tissue-specific and constitutive expression of a hypothetical gene At2g37610 in transgenic Arabidopsis

Hypothetical genes should play important roles in plant growth and development, although their biological functions await elucidation. One of these genes, namely At2g37610, caught our attention during the gene cloning of several salt-tolerant mutants. Promoter-GUS fusion analysis indicated a unique tissue-specific expression pattern of At2g37610 in Arabidopsis. Constitutive expression of the gene under 35S promoter caused obvious morphological changes in transgenic Arabidopsis plants, such as curled rosette leaves and bushy phenotype at maturity. Phenotypic characterization revealed that the cause of the bushy phenotype was the enhanced lateral bud outgrowth at the bottom region of the primary inflorescence, which is different from that of reported mutant plants (bushy or branched) such as max, axr1, and bus mutants. Together, these data suggest that At2g37610 is a possible novel gene related to the regulation of leaf development and shoot patterning.     

The nifU, nifS and nifV gene products are required for activity of all three nitrogenases of Azotobacter vinelandii

Summary Strains with mutations in 23 of the 30 genes and open reading frames in the major nif gene cluster of A. vinelandii were tested for ability to grow on N-free medium with molybdenum (Nif phenotype), with vanadium (Vnf phenotype), or with neither metal present (Anf phenotype). As reported previously, nifE, nifty, nifU, nifS and nifV mutants were Nif^– (failed to grow on molybdenum) while nifM mutants were Nif^–, Vnf^– and Anf^–. nifV, nifS, and nifU mutants were found to be unable to grow on medium with or without vanadium, i.e. were Vnf^– Anf–. Therefore neither vnf nor anf analogoues of nifU, nifS, nifV or nifM are expected to be present in A. vinelandii.     

Involvement of the rolled/MAP kinase gene in Drosophila mitosis: interaction between genes for the MAP kinase cascade and abnormal spindle

We have found that mutations that lead to loss of rolled/MAP kinase function result in a reduced mitotic index in the larval central nervous system, consistent with an interphase block to cell cycle progression, associated with a low frequency of cells showing chromosome over-condensation in mitosis and abnormal anaphase figures. In contrast to wild-type tissue, such rolled mutants do not show a significant increase in accumulation of mitotic cells when treated with colchicine. We have studied double mutant combinations between mutations affecting the activity of rolled/MAP kinase and several genes that are essential to the establishment of a bipolar spindle during progression through mitosis, and find no interactions with mutations in polo, mgr,or aurora. However, partial loss-of-function mutations in rolled enhance the abnormal spindle (asp) phenotype, whereas gain-of function mutations in rolled or in the gene encoding its activating kinase Dsor1, act as suppressors. We discuss these findings in relation to the proposed role of MAP kinase in mediating the spindle integrity checkpoint. Received: 27 October 1997 / Accepted: 18 December 1997     

cysB and cysE mutants of Escherichia coli K12 show increased resistance to novobiocin

Summary Mutations in the cysB and cysE genes of Escherichia coli K12 cause an increase in resistance to the gyrase inhibitor novobiocin but not to coumermycin, acriflavine and rifampicin. This unusual relationship was also observed among spontaneous novobiocin resistant (Nov^r) mutants: 10% of Nov^r mutants isolated on rich (LA) plates with novobiocin could not grow on minimal plates, and among those approximately half were cysB or cysE mutants. Further analyses demonstrated that cysB and cysE negative alleles neither interfere with transport of novobiocin nor affect DNA supercoiling.     

Mapping of the sor genes for l-sorbose degradation in the chromosome of Klebsiella pneumoniae

Summary A series of mutants was isolated in Klebsiella pneumoniae strain 1033, among them mutants unable to grown on l-sorbose. Different R' plasmids carrying the sor genes and other surrounding chromosomal genes were also isolated. Each plasmid contained the structural genes sorA for an Enzyme II of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system, sorD for a d-glucitol 6-phosphate dehydrogenase, sorE for an l-sorbose 1-phosphate reductase, and the corresponding regulator gene sorR. These structural genes are coordinately expressed and inducible by l-sorbose. Cis-dominant and pleiotropic mutations rendering the expression of the sor genes constitutive or eliminating it were isolated. Complementation of a series of mutations in Escherichia coli K12 and K. pneumoniae by various R' and F' plasmids and by P1 transduction in K. pneumoniae located the sor genes within the following gene sequence: rbs rha pfkA metB ppc argH ilv btuB rpoB metA ace sor pgi malB uvrA. The rbs-ilv gene loci tightly linked in E. coli K12 at 84 min, are separated in the map of K. pneumoniae 1033 and located at 86 and 89 min, respectively.     

Mutations affecting the mitochondrial genes encoding the cytochrome oxidase subunit I and apocytochrome b of Chlamydomonas reinhardtii

Mitochondrial mutants of the green alga Chlamydomonas reinhardtii that are inactivated in the cytochrome pathway of respiration have previously been isolated. Despite the fact that the alternative oxidase pathway is still active the mutants have lost the capacity to grow heterotrophically (dark + acetate) and display reduced growth under mixotrophic conditions (light + acetate). In crosses between wild-type and mutant cells, the meiotic progeny only inherit the character transmitted by the mt ^– parent, which indicates that the mutations are located in the 15.8 kb linear mitochondrial genome. Two new mutants (dum-18 and dum-19) have now been isolated and characterized genetically, biochemically and at the molecular level. In addition, two previously isolated mutants (dum-11 and dum-15) were characterized in more detail. dum-11 contains two types of deleted mitochondrial DNA molecules: 15.1 kb monomers lacking the subterminal part of the genome, downstream of codon 147 of the apocytochrome b (COB) gene, and dimers resulting from head-to-head fusion of asymmetrically deleted monomers (15.1 and 9.5 kb DNA molecules, respectively). As in the wild type, the three other mutants contain only 15.8 kb mitochondrial DNA molecules. dum-15 is mutated at codon 140 of the COB gene, a serine (TCT) being changed into a tyrosine (TAC). dum-18 and dum-19 both inactivate cytochrome c oxidase, as a result of frameshift mutations (addition or deletion of 1 bp) at codons 145 and 152, respectively, of the COX1 gene encoding subunit I of cytochrome c oxidase. In a total of ten respiratory deficient mitochondrial mutants characterized thus far, only mutations located in COB or COXI have been isolated. The possibility that the inactivation of the other mitochondrial genes is lethal for the cells is discussed.     

Isolation and characterization of temperature-sensitiveplc1 mutants of the yeastSaccharomyces cerevisiae

ThePLC1 gene of the yeastSaccharomyces cerevisiae has been discovered to encode a homolog of mammalian phosphoinositide-specific phospholipase C (PLC). Five temperature-sensitiveplc1 mutants were isolated by in vitro mutagenesis with subsequent plasmid shuffling. All of the amino acid substitutions that caused a temperature-sensitive growth phenotype were located in the X or the Y region, both of which are conserved among PLC isoenzymes. The PLC activity of all products of mutantplc1 genes was dramatically lower than that of the wild-type product, indicating that PLC activity itself is important for cell growth. At the restrictive temperature,plc1 mutant cells ceased growth at random times during the cell cycle, a result that suggests thatPLC1 is required at several or all stages of the cell cycle.     

The RCK1 and RCK2 protein kinase genes from Saccharomyces cerevisiae suppress cell cycle checkpoint mutations in Schizosaccharomyces pombe

The protein kinase-encoding genes RCK1 and RCK2 from Saccharomyces cerevisiae have been identified as suppressors of Schizosaccharomyces pombe cell cycle checkpoint mutations. Upon expression of these genes, radiation resistance is partially restored in S. pombe mutants with checkpoint deficiencies, but not in mutants with DNA repair defects. Some checkpoint mutants are sensitive to the DNA synthesis inhibitor hydroxyurea, and this sensitivity is also suppressed by RCK1 and RCK2. The degree of suppression can be modulated by varying expression levels. Expression of RCK1 or RCK2 in S. pombe causes cell elongation and decelerated growth. Cells expressing these genes have a single nucleus and a 2n DNA content. We conclude that these genes act in S. pombe to prolong the G2 phase of the cell cycle.     

Regulatory mechanisms of biosynthesis of betacyanin and anthocyanin in relation to cell division activity in suspension cultures

Regulatory mechanisms of betacyanin biosynthesis in suspension cultures of Phytolacca americana and anthocyanin in Vitis sp. were investigated in relation to cell division activity.Betacyanin biosynthesis in Phytolacca cells clearly shows a positive correlation with cell division, as the peak of betacyanin accumulation was observed at the log phase of batch cultures. Incorporation of radioactivity from labelled tyrosine into betacyanin also showed a peak at early log phase. Aphidicolin, an inhibitor of DNA synthesis, and propyzamide, an antimicrotubule drug, reduced betacyanin accumulation and inhibited the incorporation of radioactivity from labelled tyrosine into betacyanin at concentrations which were inhibitory to cell division. Both inhibitors reduced the incorporation of radioactivity from labelled tyrosine to 3,4-dihydroxyphenylalanine (DOPA), but the incorporation of labelled DOPA into betacyanin was not affected. These results suggest that the conversion of tyrosine to DOPA is coupled with cell division activity.In contrast, the anthocyanin accumulation in Vitis cells showed a negative correlation with cell division. Accumulation occurred at the stationary phase in batch cultures when cell division ceased. Aphidicolin or reduced phosphate concentration induced a substantial increase in anthocyanin accumulation as well as the inhibition of cell division. Chalcone synthase (CHS) activity increased at the time of anthocyanin accumulation. Northern blotting analysis indicated that changes in CHS mRNA levels corresponded to similar changes in enzymatic activity. The pool size of endogenous phenylalanine was low during active cell division, but increased before anthocyanin began to accumulate and concomitantly with increasing levels of CHS mRNA. Exogenous supply of phenylalanine at the time of low endogenous levels induced the elevation of CHS mRNA and anthocyanin accumulation. These results indicate that the elevation of endogenous phenylalanine levels, when cell division ceases, may cause the increase in CHS mRNA levels, resulting in increased CHS activity and subsequently in anthocyanin accumulation in Vitis suspension cultures.Abbreviations CHS chalcone synthase - CHFI chalcone flavanone isomerase - DOPA 3,4-dihydroxyphenylalanine - PAL phenylalanine ammonia lyase     

Functional expression of Aquaspirillum magnetotacticum genes in Escherichia coli K12

Summary Gene libraries from the magnetotactic bacterium, Aquaspirillum magnetotacticum were constructed in Escherichia coli with cosmids pLAFR3 and c2RB as vectors. Recombinant cosmids able to complement the thr-1, leuB, and proA mutations of the host were identified. The Pro⁺ recombinant cosmid restored wild-type phenotype in proA and proB but not in the proC mutants of E. coli. The results of restriction endonuclease digestion and Southern hybridization analysis indicate that the relevent leu and pro biosynthetic genes of A. magnetotacticum are not closely linked on the chromosome.