RESUMO
In plants, many invading microbial pathogens are recognized by cell-surface pattern recognition receptors, which induce defense responses. Here, we show that the ceramide Phytophthora infestans-ceramide D (Pi-Cer D) from the plant pathogenic oomycete P. infestans triggers defense responses in Arabidopsis. Pi-Cer D is cleaved by an Arabidopsis apoplastic ceramidase, NEUTRAL CERAMIDASE 2 (NCER2), and the resulting 9-methyl-branched sphingoid base is recognized by a plasma membrane lectin receptor-like kinase, RESISTANT TO DFPM-INHIBITION OF ABSCISIC ACID SIGNALING 2 (RDA2). 9-Methyl-branched sphingoid base is specific to microbes and induces plant immune responses by physically interacting with RDA2. Loss of RDA2 or NCER2 function compromised Arabidopsis resistance against an oomycete pathogen. Thus, we elucidated the recognition mechanisms of pathogen-derived lipid molecules in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ceramidas , Interações Hospedeiro-Patógeno , Ceramidase Neutra , Phytophthora infestans , Doenças das Plantas , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ceramidas/metabolismo , Ceramidase Neutra/genética , Ceramidase Neutra/metabolismo , Phytophthora infestans/patogenicidade , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Mitogênicos/genética , Receptores Mitogênicos/metabolismoRESUMO
Ehrlich ascites tumor cell extracts form a gel when warmed to 25 degrees C at pH 7.0 in sucrose solution, and the gel rapidly becomes a sol when cooled to 0 degrees C. This gel-sol transformation was studied quantitatively by determining the volume or the total protein of pellets of gel obtained by low-speed centrifugation. The gelation depended on nucleotide triphosphates, Mg2+, KCl, and a reducing agent. Gelation was inhibited reversibly by 0.5 microM free Ca2+, and 25--50 ng/ml of either cytochalasin B or D, but it was not affected by 10 mM colchicine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the gel was composed of six major proteins with mol wt greater than 300,000, 270,000, 89,000, 51,000, 48,000, and 42,000 daltons. The last component was identified as cell actin because it had the same molecular weight as muscle actin and bound with muscle myosin and tropomyosin. The role of actin in gelation was studied by use of actin-inhibitors. Gelation was inhibited by a chemically modified subfragment-1 of myosin, which binds with F-actin even in the presence of ATP, and by bovine pancreatic DNase I, which tightly binds with G-actin. Muscle G-actin neutralized the inhibitory effect of DNase I when added at an equimolar ratio to the latter, and it also restored gelation after its inhibition by DNase I. These findings suggest that gelation depends on actin. However, the extracts showed temperature-dependent, cytochalasin-sensitive, and Ca2+-regulated gelation as did the original extracts when the cell actin in the extracts was replaced by muscle actin, suggesting that components other than cell actin might be responsible for these characteristics of the gelation.
Assuntos
Actinas , Carcinoma de Ehrlich , Extratos de Tecidos , Actinas/análise , Animais , Cálcio/farmacologia , Fenômenos Químicos , Química , Citocalasina B/farmacologia , Desoxirribonucleases/farmacologia , Géis , Camundongos , Miosinas/farmacologia , Proteínas de Neoplasias/análise , Nucleotídeos/farmacologia , Temperatura , Extratos de Tecidos/análiseRESUMO
The long-standing supposition that the biological clock cannot function in cells that divide more rapidly than the circadian cycle was investigated. During exponential growth in which the generation time was 10 hours, the profile of bioluminescence from a reporter strain of the cyanobacterium Synechococcus (species PCC 7942) matched a model based on the assumption that cells proliferate exponentially and the bioluminescence of each cell oscillates in a cosine fashion. Some messenger RNAs showed a circadian rhythm in abundance during continuous exponential growth with a doubling time of 5 to 6 hours. Thus, the cyanobacterial circadian clock functions in cells that divide three or more times during one circadian cycle.
Assuntos
Ritmo Circadiano , Cianobactérias/fisiologia , Divisão Celular , Cianobactérias/citologia , Cianobactérias/genética , Cianobactérias/crescimento & desenvolvimento , Genes Reporter , Luciferases/genética , Luciferases/metabolismo , Luminescência , Mutação , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteína do Fotossistema II , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transformação BacterianaRESUMO
A diverse set of circadian clock mutants was isolated in a cyanobacterial strain that carries a bacterial luciferase reporter gene attached to a clock-controlled promoter. Among 150,000 clones of chemically mutagenized bioluminescent cells, 12 mutants were isolated that exhibit a broad spectrum of periods (between 16 and 60 hours), and 5 mutants were found that show a variety of unusual patterns, including arrhythmia. These mutations appear to be clock-specific. Moreover, it was demonstrated that in this cyanobacterium it is possible to clone mutant genes by complementation, which provides a means to genetically dissect the circadian mechanism.
Assuntos
Ritmo Circadiano/genética , Cianobactérias/genética , Genes Bacterianos , Clonagem Molecular , Cianobactérias/crescimento & desenvolvimento , Cianobactérias/fisiologia , Escuridão , Teste de Complementação Genética , Luz , Medições Luminescentes , Mutagênese , Mutação , TemperaturaRESUMO
Cyanobacteria are the simplest organisms known to have a circadian clock. A circadian clock gene cluster kaiABC was cloned from the cyanobacterium Synechococcus. Nineteen clock mutations were mapped to the three kai genes. Promoter activities upstream of the kaiA and kaiB genes showed circadian rhythms of expression, and both kaiA and kaiBC messenger RNAs displayed circadian cycling. Inactivation of any single kai gene abolished these rhythms and reduced kaiBC-promoter activity. Continuous kaiC overexpression repressed the kaiBC promoter, whereas kaiA overexpression enhanced it. Temporal kaiC overexpression reset the phase of the rhythms. Thus, a negative feedback control of kaiC expression by KaiC generates a circadian oscillation in cyanobacteria, and KaiA sustains the oscillation by enhancing kaiC expression.
Assuntos
Proteínas de Bactérias/genética , Relógios Biológicos/genética , Ritmo Circadiano/genética , Cianobactérias/genética , Regulação Bacteriana da Expressão Gênica , Sequência de Aminoácidos , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano , Clonagem Molecular , Cianobactérias/fisiologia , Retroalimentação , Genes Bacterianos , Genes Reporter , Luminescência , Modelos Biológicos , Dados de Sequência Molecular , Família Multigênica , Mutação , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão , Transcrição GênicaRESUMO
Recombinant phage particles carrying the thymidine kinase (TK) gene of herpes simplex virus type 1, coprecipitated with calcium phosphate, efficiently transformed mouse Ltk- cells to the TK+ phenotype. The conditions necessary to achieve high efficiency of transfer of the TK gene by phage particle-mediated gene transfer were investigated. Of the parameters examined, the pH of the buffer used for coprecipitation of phage particles with calcium phosphate, the length of time of coprecipitation, and the length of the adsorption period were found to alter the transfer efficiency significantly. The optimal pH was 6.87 at 25 degrees C. The other optimal values for these parameters were as follows: coprecipitation time, 7 to 20 min; adsorption time, 18 to 30 h. Treatment with dimethyl sulfoxide, glycerol, or sucrose did not enhance gene transfer. The optimal conditions yielded about 1 transformant per 10(5) phage particles per 10(6) cells without carrier DNA. An increase in the dosage of phage particles, up to at least 5 x 10(7) phage particles per 100-mm dish, resulted in a linear increase in the number of transformants. Addition of carrier phage, up to 10(10) phage particles per dish, did not significantly affect the number of transformants.
Assuntos
Bacteriófagos/genética , Herpesvirus Humano 1/genética , Timidina Quinase/genética , Transfecção/métodos , Adsorção/efeitos dos fármacos , Animais , Bacteriófagos/fisiologia , Fosfatos de Cálcio , Contagem de Células , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Transformação Celular Viral , Precipitação Química , Dimetil Sulfóxido/farmacologia , Glicerol/farmacologia , Herpesvirus Humano 1/enzimologia , Concentração de Íons de Hidrogênio , Camundongos , Plasmídeos/genética , Mapeamento por Restrição , Sacarose/farmacologia , Fatores de TempoRESUMO
Photolyase is a DNA repair enzyme that reverses UV-induced photoproducts in DNA in a light-dependent manner. Recently, photolyase homologs were identified in higher eukaryotes. These homologs, termed crypto-chromes, function as blue light photoreceptors or regulators of circadian rhythm. In contrast, most bacteria have only a single photolyase or photolyase-like gene. Unlike other microbes, the chromosome of the cyanobacterium SYNECHOCYSTIS: sp. PCC6803 contains two ORFs (slr0854 and sll1629) with high similarities to photolyases. We have characterized both genes. The slr0854 gene product exhibited specific, light-dependent repair activity for a cyclo-butane pyrimidine dimer (CPD), whereas the sll1629 gene product lacks measurable affinity for DNA in vitro. Disruption of either slr0854 or sll1629 had little or no effect on the growth rate of the cyanobacterium. A mutant lacking the slr0854 gene showed severe UV sensitivity, in contrast to a mutant lacking sll1629. Phylogenetic analysis showed that sll1629 is more closely related to the cryptochromes than photolyases. We conclude that sll1629 is a bacterial cryptochrome. To our knowledge, this is the first description of a bacterial cryptochrome.
Assuntos
Cianobactérias/genética , DNA Bacteriano/efeitos da radiação , Desoxirribodipirimidina Fotoliase/genética , Proteínas de Drosophila , Proteínas do Olho , Flavoproteínas/genética , Células Fotorreceptoras de Invertebrados , Raios Ultravioleta , Sequência de Bases , Criptocromos , Cianobactérias/crescimento & desenvolvimento , Cianobactérias/efeitos da radiação , Reparo do DNA , DNA Bacteriano/genética , Escuridão , Genes Bacterianos , Luz , Dados de Sequência Molecular , Filogenia , Receptores Acoplados a Proteínas GRESUMO
Recombinant Charon 4A phages accommodating the Herpes simplex virus (HSV-1) thymidine kinase (tk) gene, the ampicillin-resistance (ApR) gene, and the replication origin of pBR322 were constructed. The phage DNA was introduced into mouse Ltk- cells by a free DNA transfer method or phage-mediated DNA transfer method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607]. Analyses of the physical state of the transferred DNA in the recipient cell genome showed that a DNA fragment as long as 12.7 kb was integrated intact into 67% and less than 40% of the Ltk- transformant cells by phage-mediated DNA transfer and by free DNA transfer, respectively. We also developed a new rapid method for recovery of the transferred gene from the Ltk+ cell into Escherichia coli; the method depends on the fact that the recombinant lambda phage carrying the ApR gene and replication origin of pBR322 transduces lambda-lysogenic bacteria to ApR and is maintained as a plasmid. Using this method the HSV-1 tk gene from one Ltk+ transformant was rapidly and successfully recovered without any rearrangement of the target sequence.
Assuntos
Bacteriófago lambda/genética , Vetores Genéticos , Plasmídeos , Animais , DNA Recombinante , Células L , Camundongos , Simplexvirus/genética , Timidina Quinase/genéticaRESUMO
An efficient procedure for the introduction of recombinant cosmids into cultured mammalian cells consists of the following steps. Cosmids were packaged, in vitro, into lambda phage particles and transduced into Escherichia coli hosts lysogenized with thermo-inducible lambda c Its phage. The introduced cosmids were repackaged into phage particles in the thermo-induced hosts. The efficiency of such in vivo cosmid packaging was further improved by construction of pTC vectors that carried three cohesive end sites (cos) of phage lambda, arrayed in tandem. Two types of cosmids, in almost equal numbers (i.e., cosmids with one cos and cosmids with two cos), were obtained from a cosmid library constructed with pTC vectors. The efficiency of packaging in vivo of cosmids with two cos, was found to be 7-20 times higher than that of corresponding cosmids with only one cos. Use of a high-copy-number derivative of pTCl further improved the phage yield by 20- to 30-fold. The packaged cosmids, which carried the thymidine kinase-encoding gene of herpes simplex virus type 1 as a selective marker, were introduced into mouse Ltk- cells with an efficiency of 10(-5), by the phage transfer method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607-616].
Assuntos
Clonagem Molecular/métodos , Cosmídeos , Animais , Bacteriófago lambda/genética , Células L , Camundongos , Proteínas Recombinantes/genética , Timidina Quinase/genéticaRESUMO
We constructed a series of cosmid vectors that carry two tandemly arranged lambda cos and mammalian selective markers. We achieved cloning efficiencies of 1-3 x 10(7) and greater than 10(6) colony-forming units per microgram of insert, using a cloned 42-kb BamHI fragment and Sau3AI fragments of 40-50 kb from mouse genomic DNA, respectively. The modified Ca.phosphate coprecipitation method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607-616] considerably improved the efficiency of gene transfer of cosmids into cultured mammalian cells: when genes encoding thymidine kinase from herpes simplex virus type 1 and aminoglycoside 3'-phosphoribosyltransferase from Tn5 were selected, the efficiencies of gene transfer into mouse L cells were about 10(-6). The mouse genome contains one copy of the functional gene for elongation factor 2 (EF2) per haploid genome and multiple copies of the EF2-related gene. We isolated a cosmid that carried functional full-length mouse EF2 from a cosmid library of L-cell genomic DNA, by colony hybridization and subsequent gene transfer of candidate cosmids into human 143B cells.
Assuntos
Cosmídeos , Genes , Vetores Genéticos , Fatores de Alongamento de Peptídeos/genética , Transfecção , Animais , DNA/genética , DNA/isolamento & purificação , Biblioteca Gênica , Células L/metabolismo , Camundongos , Fator 2 de Elongação de Peptídeos , Fosfoproteínas/genética , Mapeamento por RestriçãoRESUMO
We cloned a full-length complementary DNA for the hemagglutinin-neuraminidase (HN) mRNA of Sendai virus (HVJ) using a synthetic 27-mer as a probe. Nucleotide sequence analysis showed that there is a long open reading frame on the mRNA that encodes a protein of 575 amino acids. The deduced amino acid sequence indicated that only one hydrophobic region sufficiently long to anchor the protein in the membrane and located near the N-terminus (amino acids 35-60). It is suggested that HN protein is oriented with its N-terminus inside the membrane.
Assuntos
Clonagem Molecular , DNA/genética , Vírus da Parainfluenza 1 Humana/genética , RNA Mensageiro/genética , RNA Viral/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Recombinante , Proteína HN , Vírus da Parainfluenza 1 Humana/enzimologiaRESUMO
We analyzed a male patient with Fabry's disease who had no activity of the lysosomal hydrolase alpha-galactosidase A (alpha-GalA) and female members of his family. We cloned a cDNA that encoded the mutant alpha-GalA, determined its nucleotide sequence, and found two nucleotide differences between the mutant and the wild-type cDNAs. Although one difference was silent, the other difference, a C-to-T transition at nucleotide number 118, resulted in an amino acid substitution of Pro-40 by Ser. A transient expression assay demonstrated that this missense mutation was the cause of the deficiency of alpha-GalA activity in the patient. In vitro mutagenesis experiments demonstrated that Pro-40 is critical for the appearance of alpha-GalA activity.
Assuntos
Doença de Fabry/enzimologia , Galactosidases/genética , Prolina , Serina , alfa-Galactosidase/genética , Sequência de Bases , Northern Blotting , Células Cultivadas , DNA/genética , Doença de Fabry/genética , Humanos , Lisossomos/enzimologia , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Conformação Proteica , Pele/enzimologia , alfa-Galactosidase/metabolismoRESUMO
kaiABC, a gene cluster, encodes KaiA, KaiB and KaiC proteins that are essential to circadian rhythms in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. Kai proteins can interact with each other in all possible combinations. This study identified two KaiA-binding domains (C(KABD1) and C(KABD2)) in KaiC at corresponding regions of its duplicated structure. Clock mutations on the two domains and kaiA altered the strength of C(KABD)-KaiA interactions assayed by the yeast two-hybrid system. Thus, interaction between KaiA and KaiC through C(KABD1) and C(KABD2) is likely important for circadian timing in the cyanobacterium.
Assuntos
Proteínas de Bactérias/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Ritmo Circadiano , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano , Glutationa Transferase/metabolismo , Modelos Biológicos , Modelos Moleculares , Família Multigênica , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Human alpha-3-N-acetylgalactosaminyltransferase has been purified 27,000,000-fold from A1 plasma by (NH4)2SO4 fractionation and affinity chromatography on Sepharose 4B, anti-human group O plasma antibodies-Sepharose 4B, and Blue Dextran-Sephadex G-25. A modified procedure in the Sepharose 4B step was developed by batch adsorption and desorption experiments. Cibacron Blue F3G-A, the chromophore of Blue Dextran, was found to bind to the enzyme. UDP is an effective inhibitor of this binding. The pure transferase has an apparent molecular weight of 35,000 as judged by SDS-PAGE in the presence of a reducing agent. The specific activity is 16 pmol/min.ng enzyme, which is comparable to that (30 pmol/min.ng enzyme) of alpha-3-N-acetylgalactosaminyltransferase from porcine submaxillary glands [Schwyzer and Hill (1977) J. Biol. Chem. 252, 2338-2355]. The apparent Km values for UDP-GalNAc, 2'-fucosyllactose, and lacto-N-fucopentaose I are 13, 270, and 350 microM, respectively. The reaction velocity was found to fall off again at high concentrations of oligosaccharide acceptor substrates. The apparent Ki values for UDP and UDP-galactose are 8.6 and 6.2 microM, respectively. The pure enzyme also catalyzes the transfer of galactose in alpha-linkage to 2'-fucosyllactose though the transfer rate of galactose is much lower than that of N-acetylgalactosamine.
Assuntos
Sistema ABO de Grupos Sanguíneos/genética , Hexosaminidases/genética , Soluções Tampão , Fenômenos Químicos , Físico-Química , Cromatografia de Afinidade , Cromatografia em Gel , Estabilidade de Medicamentos , Eletroforese em Gel de Poliacrilamida , Hexosaminidases/isolamento & purificação , Hexosaminidases/metabolismo , Humanos , Imunoglobulina G/análise , Imunoglobulina G/isolamento & purificação , Cinética , Manganês/análise , Peso Molecular , Difosfato de Uridina/metabolismo , Uridina Difosfato Galactose/metabolismo , alfa-N-AcetilgalactosaminidaseRESUMO
Recent progress in techniques for the transfer of genes into cultured mammalian cells has made possible the isolation of various interesting genes from other mammalian cells, and also the study of the function and the regulation of gene expression of mammalian genes in vivo. Various transfection techniques have been developed: incubation of recipient cells with genes in the presence of diethylaminoethyldextran (see Chapter 3 , this vol.; ref. 1); incubation of the cells with metaphase chromosomes and poly-L-ornithine (2); and incubation of the cells with coprecipitates of calcium phosphate and genetic material, such as DNA ( Chapter 2 ; refs. 3,4), metaphase chromosomes ( Chapter 10 ; ref. 5), and λ phage particles (6,7). Other methods for gene transfer have also been developed: direct injection of DNA into the nuclei of recipient cells (8,9); use of viral vectors ( Chapter 11 - Chapter 15 ; for review, see also ref. 10); fusion of the recipient cells with bacterial spheroplasts (11) or liposomes (10-14); and fusion of cells, mediated by HVJ (Sendai virus), with liposomes (15-17) or reconstituted erythrocyte membrane vesicles (18). The cell membranes of recipient cells have also been rendered permeable to genetic material by electric pulses ( Chapter 5 ; ref. 19) Chapter 11 Chapter 13 15; for review, see also ref. 10); fusion of the recipient cells with bacterial spheroplasts (11) or liposomes (10-14); and fusion of cells, mediated by HVJ (Sendai virus), with liposomes (15-17) or reconstituted erythrocyte membrane vesicles (18). The cell membranes of recipient cells have also been rendered permeable to genetic material by electric pulses ( Chapter 5 ; ref. 19).
RESUMO
A Japanese male patient with Fabry disease who had no activity of the lysosomal hydrolase alpha-galactosidase A (alpha-GalA) and female members of his family were analyzed. We cloned a cDNA encoded the mutant alpha-GalA, determined its nucleotide sequence, and found two nucleotide differences between the mutant and the wild-types cDNAs. The one difference, a C-to-T transition at nucleotide number 118, resulted in an amino acid substitution of Pro-40 by Ser. A transient expression assay demonstrated that this missense mutation was the cause of the deficiency of alpha-GalA activity in the patient. Gene analysis of the patient's family by PCR and subsequent sequencing demonstrated that all females were heterozygotes.
Assuntos
Doença de Fabry/genética , alfa-Galactosidase/genética , Adulto , Povo Asiático/genética , Sequência de Bases , DNA , Análise Mutacional de DNA , Doença de Fabry/etiologia , Feminino , Heterozigoto , Humanos , Japão , Masculino , Mutação , Reação em Cadeia da PolimeraseAssuntos
Actomiosina/metabolismo , Músculos , Miosinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Actinas/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Cálcio/metabolismo , Precipitação Química , Cloromercurobenzoatos , Rim/enzimologia , Proteínas Musculares/metabolismo , Physarum , Potássio/metabolismo , Coelhos , Retículo Sarcoplasmático/metabolismo , Sódio/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Reproducible circadian rhythms of bioluminescence from individual colonies of cyanobacteria (Synechococcus sp. strain PCC 7942) has been observed. Phenotypic monitoring of colonies on agar plates will enable us to genetically analyze the molecular mechanism of the circadian clock of cyanobacteria by screening for clock mutants. By the introduction of a bacterial luciferase gene, we previously developed a transformed cyanobacterial strain (AMC149) that expresses luciferase as a bioluminescent reporter of the circadian clock. In liquid culture, AMC149 expresses a rhythm of bioluminescence that displays the same behavior as circadian rhythms in higher eukaryotes. Improvements in the technique for administering the reporter enzyme's substrate (decanal) and a highly sensitive photon-counting camera allow monitoring the bioluminescence of single colonies. Individual colonies on agar plates displayed a rhythmicity which is essentially the same as that previously reported for liquid cultures.
Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Cianobactérias/fisiologia , Medições Luminescentes , Cianobactérias/isolamento & purificação , Genes Reporter , Luciferases/genética , Luciferases/metabolismo , Fótons , Proteínas Recombinantes/metabolismo , Transformação Genética , Vibrio/enzimologia , Vibrio/genéticaRESUMO
A circadian clock, with physiological characteristics similar to those of eukaryotes, functions in the photosynthetic prokaryote, cyanobacteria. The molecular mechanism of this clock has been efficiently dissected using a luciferase reporter gene that reports the status of the clock. A circadian clock gene cluster, kaiABC, has been cloned via rhythm mutants of cyanobacterium, Synechococcus, and many clock mutations mapped to the three kai genes. Although kai genes do not share any homology with clock genes so far identified in eukaryotes, analysis of their expression suggests that a negative feedback control of kaiC expression by KaiC generates the circadian oscillation and that KaiA functions as a positive factor to sustain this oscillation. BioEssays 22:10-15, 2000.