Characterization of a Non-abscission Mutant in Lupinus angustifolius L.: Physiological Aspects

A single-gene recessive mutant (Abs^-) of Lupinus angustifoliusL. ‘Danja’ that does not abscise any organs wascompared with its parent during continuous exposure of explantsfrom 14 d old seedlings to 10 µl l^-1ethylene. Both endo-(1,4)-ß- D -glucanase (cellulase) and polygalacturonase(PGA) activities increased significantly and progressively inpetiole-stem abscission zones of the parent before the onsetof abscission, and were reflected in a rapid decline in breakstrengthfrom 300 to 70 g within 32 h. In the mutant there was negligibleincrease in hydrolytic enzyme activity, breakstrength declinedslowly (to 180–200 g by 72 h) and there was no abscission.Isoelectric focusing showed two cellulase isoforms (pI 5.0 andpI 8.5) expressed in abscission zones of the parent; these wereexpressed at much lower levels in the mutant. These data areinterpreted to indicate that expression of at least two formsof cellulase activity is enhanced by ethylene in normal petioleabscission zones of lupin. PGA activity also increased in theabscission zone tissue of the parent but to a lesser extentin that of the mutant. We attribute the Abs^-phenotype to mutationof a gene regulating ethylene-responsive expression of abscission-specifichydrolytic enzymes. Copyright 2001 Annals of Botany Company Lupinus angustifolius, abscission, breakstrength, cellulase, ethylene, legume, lupin, mutant, polygalacturonase     

The Effect of thecrp Genotypes on the Transformation Efficiency in Escherichia coli

We have found that a significant difference exists in transformationefficiency between the crp⁺/crp^– isogenic pair of strainsof Escherichia coli, with the efficiency being much higher incrp^– than in crp⁺. The ratio of transformation efficiencybetween crp⁺ and crp^– strains depends very little on theplasmid size. This observation suggests that the differenceof the transformation efficiency is due to mechanisms otherthan a crp-regulated endonuclease. The crp gene is one of thefirst specific genes that have been shown to affect transformationefficiency.     

A Role for the acrA Gene in the Protection of Escherichia coli Cells against Excess Sodium

The acrA gene determines the sensitivity of Escherichia coliK-12 cells to acriflavine, which is one of the acridine dyesand which effectively eliminates certain plasmids from the bacterialcells. The acriflavine-sensitivity mutation leads to instabilityof plasmids, such as sex (F)- and drug-resistance (R)-factorsand to loss of a membrane protein with molecular weight about60 kDa (Nakamura 1974, 1976, Nakamura et al. 1975, 1981). Wehave found that cells with a mutant acrA gene were also moresensitive to an excess of sodium ions in the medium than werethe wild-type acrA⁺ cells (Nakamura 1977). The product of theacrA gene hindered the accumulation of sodium by the cells.Although mutations in theproA or proB genes, which determinethe synthesis of the enzymes y-glutamyl phosphate reductaseand y-glutamyl kinase, respectively, in the proline-biosyntheticpathway also led to sensitivity to and accumulation of excesssodium, the presence of the acrA⁺ allele decreased both theseparameters. (Received November 1, 1989; Accepted April 27, 1990)     

A Mutant of Arabidopsis thaliana That Defines a New Locus for Glycine Decarboxylation

A novel photorespiratory mutant of Arabidopsis thaliana, designatedgld2, was isolated based on a growth requirement for abnormallyhigh levels of atmospheric CO₂. Photosynthetic CO₂ fixationwas inhibited in the mutant following illumination in air butnot in atmosphere containing 2% O₂. Photosynthetic assimilationof ¹⁴CO₂ in an atmosphere containing 50% O₂ resulted in accumulationof 48% of the soluble label in glycine in the mutant comparedto 9% in the wild type. The rate of glycine decarboxylationby isolated mitochondria from the mutant was reduced to 6% ofthe wild type rate. In genetic crosses, the mutant complementedtwo previously described photorespiratory mutants of A. thalianathat accumulate glycine during photosynthesis in air due todefects in glycine decarboxylase (glyD, now designated gld1)and serine transhydroxymethylase (stm). Because glycine decarboxylaseis a complex of four enzymes, these results are consistent witha mutation in a glycine decarboxylase subunit other than thataffected in the gld1 mutant. The two gld loci were mapped tochromosomes 2 and 5, respectively. ³Present address: Department of Crop and Soil Sciences, MichiganState University, East Lansing, MI 48824, U.S.A. ⁴Present address: Department of Applied Bioscience, Facultyof Agriculture, Hokkaido University, Kita-Ku, Sapporo, 060 Japan ⁵Present address: Department of Biology, Carnegie Institutionof Washington, 290 Panama Street, Standford, CA 94305, U.S.A.     

Functional Analysis of Fructose-1,6-Bisphosphatase Isozymes (fbp-I and fbp-II Gene Products) in Cyanobacteria

Synechococcus PCC 7942 contains two fructose-1,6-bisphosphataseisozymes (FBPase-I and FBPase-II), while Synechocystis PCC 6803has only one (FBPase-I) in spite of the occurrence of two FBPaseisozyme genes [Tamoi et al. (1998) Biochim. Biophys. Acta 1383:232]. We now demonstrate that disruption of the gene encodingFBPase-II (fbp-II) with a kanamycin resistance gene cartridgedoes not affect cell growth, Chl content, or CO² assimilationin Synechococcus PCC 7942, and disruption of the gene encodingFBPase-I (fbp-I) is a lethal mutation in both cyanobacteria.Accordingly, it is clear that FBPase-I is necessary to sustainphotosynthesis and gluconeogenesis in cyanobacteria. (Received September 10, 1998; Accepted December 10, 1998)     

Maternal Effects of mto1 Mutation, That Causes Overaccumulation of Soluble Methionine, on the Expression of a Soybean {beta}Conglycinin Gene Promoter-GUS Fusion in Transgenic Arabidopsis thaliana

ß-Conglycinin, the 7S seed storage protein of soybean(Glycine max [L.] Merr.), is comprised mainly of three subunits,designated , ' and ß. Expression of the gene encodingthe ß subunit is unique because its expression hasbeen shown to be down-regulated by exogenously applied L-methioninein immature soybean cotyledon cultures in vitro. Arabidopsisthaliana strain carrying a mto1-1 mutation overaccumulates solublemethionine. By using this mutant, we analyzed the effects ofmethionine on expression of the ß subunit gene invivo. Reciprocal crosses were made between the mto1-1 mutantand a transgenic A. thaliana strain, designated SNTß3,which carries a ß-glucuronidase (GUS) reporter geneunder the control of the promoter region of the ßsubunit gene. Analysis of GUS activity in F₁ seeds indicatedthat the GUS activity was dramatically repressed when the mto1-1mutant plants were used as female parents. We constructed astrain which carries both the transgene and mto1-1 mutationin the homozygous state. Analyses of the GUS activity in seedsof this double homozygous strain indicated that the GUS activitywas repressed to 2.5% of control by introduction of the mto1-1mutation. These results indicate that the ß subunitgene promoter activity in seeds is down-regulated by maternalgenotype and suggest that soluble methionine, or its mobilemetabolite, is translocated from mother plants to repress ßsubunit gene expression in seeds. ⁵Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan ⁶Present address: Department of Biotechnology, Faculty of Agriculture,The University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan     

Modulation of chloride secretory responses and barrier function of intestinal epithelial cells by the Salmonella effector protein SigD

The Salmonella effector protein SigD is an inositol phosphate phosphatase that inhibits phosphatidylinositol 3-kinase-dependent signaling. Because epidermal growth factor (EGF) inhibits chloride secretion via phosphatidylinositol 3-kinase, we explored whether Salmonella infection might modify the inhibitory effect of EGF. As expected, EGF inhibited chloride secretion induced by carbachol in T₈₄ epithelial cells. Infection with wild-type (WT) but not sigD^– mutant S. typhimurium SL1344 decreased CCh-stimulated chloride secretion. Moreover, WT but not sigD^– Salmonella reduced the inhibitory effect of EGF on carbachol-stimulated chloride secretion. Complementation of sigD restored the ability of mutant Salmonella to reverse the inhibitory effect of EGF. EGF-induced EGF receptor phosphorylation was similar in cells infected with either WT or mutant Salmonella, and neither WT nor sigD^– Salmonella altered recruitment of the p85 subunit of phosphatidylinositol 3-kinase to EGF receptor, implying that SigD acts downstream of these signaling events. Furthermore, transepithelial resistance fell more rapidly in cells infected with WT vs. sigD^– Salmonella, indicating an early role for SigD in reducing barrier function, perhaps via activation of protein kinase C. We conclude that the Salmonella bacterial effector protein SigD may play critical roles in the pathogenesis of disease caused by this microorganism. chloride secretion; Salmonella typhimurium; epidermal growth factor     

Formation of a series of myo-inositol phosphates during growth of rice plant cells in suspension culture

A series of myo-inositol phosphates including myo-inositol mono-to hexa-phosphates was observed during growth of cultured riceplant cells. We also found that ³²Pi and myo-[2-³H] inositolwere incorporated into all these myo-inositol phosphates. myo-Inositolphosphorylating activity, which depended on ATP and Mg²⁺, wasdetected in the soluble fraction from the cells, and the reactionproduct was identified as myo-inositol-2-phosphate. (Received January 21, 1980; )     

The Effect of Mutations in the T-DNA Encoded Auxin Pathway on the Endogenous Phytohormone Content in Cloned Nicotiana tabacum Crown Gall Tissues

We analyzed the endogenous auxin and cytokinin levels of clonedNicotiana tabacum SR 1-lines induced either by the wild-typeAgrobacterium tumefaciens C58 strain or by mutants affectedin the T-DNA-encoded IAA biosynthesis pathway. The wild-typeSR1-C58 line contained up to 20 times more IAA than a nontransformedSRI-callus line. The mutant lines affected in gene 1 (iaaM^–)or gene 2 (iaaH^–) contained intermediate levels of IAA. Analysis of the endogenous levels of indole-3-acetamide (IAM)in the nontransformed SR 1 callus line, the wild-type SR1-C58and the two mutant lines confirmed the T-DNA-induced IAA biosynthesispathway in the transformed tumor cells. Supplementing auxinto the mutant lines resulted in complete suppression of theshoot-forming ability, but no changes in the endogenous IAAlevels. There was no marked difference in the cytokinin level betweenthe nontransformed callus line and the wild type tumor line.The two mutant lines, however, showed a 20- to 30-fold highercytokinin level which was not affected by the addition of NAA.The T-DNA encoded hormone biosynthetic pathways are discussedin relation to pathways of the host plant. (Received July 29, 1986; Accepted February 14, 1987)     

Procera is a putative DELLA mutant in tomato (Solanum lycopersicum): effects on the seed and vegetative plant

The procera (pro) mutant of tomato exhibits a well-characterizedconstitutive gibberellic acid (GA) response phenotype. The tomatoDELLA gene LeGAI in the pro mutant background contains a pointmutation that results in an amino acid change in the conservedVHVID putative DNA-binding domain in LeGAI to VHEID. This samepoint mutation is in four different genetic backgrounds exhibitingthe pro phenotype, suggesting that this mutation co-segregateswith the pro phenotype. Complementation of the mutant with aconstitutively expressed wild-type LeGAI gene sequence was notconclusive due to the infertility of transgenic plants. Thepro mutation alters tomato branching architecture through differentialsuppression of axillary bud development, indicating a role forDELLA proteins in the regulation of plant structure. Isolatedgib-1 pro double mutant embryo axes, which are unable to synthesizeGA, germinate faster than their wild-type counterparts, andexert greater embryo growth potential. The pro mutation is thereforeregulating GA responses within the tomato embryo. Transientexpression of a LeGAI–GFP (green fluorescent protein)fusion protein in onion epidermis results in its location tothe nucleus, and this protein is rapidly degraded by the proteasomein the presence of GA. Key words: Branching pattern, DELLA, embryo growth potential, tomato seed germination Received 12 October 2007; Revised 27 November 2007 Accepted 28 November 2007     

The mitochondrial external NADPH dehydrogenase modulates the leaf NADPH/NADP+ ratio in transgenic Nicotiana sylvestris

Plant mitochondria contain alternative external NAD(P)H dehydrogenases,which oxidize cytosolic NADH or NADPH and reduce ubiquinonewithout inherent linkage to proton pumping and ATP production.In potato, St-NDB1 is an external Ca²⁺-dependent NADPH dehydrogenase.The physiological function of this enzyme was investigated inhomozygous Nicotiana sylvestris lines overexpressing St-ndb1and co-suppressing St-ndb1 and an N. sylvestris ndb1. In leafmitochondria isolated from the overexpressor lines, higher activityof alternative oxidase (AOX) was detected. However, the AOXinduction was substantially weaker than in the complex I-deficientCMSII mutant, previously shown to contain elevated amounts ofNAD(P)H dehydrogenases and AOX. An aox1b and an aox2 gene wereup-regulated in CMSII, but only aox1b showed a response, albeitsmaller, in the transgenic lines, indicating differences inAOX activation between the genotypes. As in CMSII, the increaseof AOX in the overexpressing lines was not due to a generaloxidative stress. The lines overexpressing St-ndb1 had consistentlylowered leaf NADPH/NADP⁺ ratios in the light and variably decreasedlevels in darkness, but unchanged NADH/NAD⁺ ratios. CMSII insteadhad similar NADPH/NADP⁺ and lower NADH/NAD⁺ ratios than thewild type. These results demonstrate that St-NDB1 is able tomodulate the cellular balance of NADPH and NADP⁺ at least inthe day and that reduction of NADP(H) and NAD(H) is independentlycontrolled. Similar growth rates, chloroplast malate dehydrogenaseactivation and xanthophyll ratios indicate that the change inreduction does not communicate to the chloroplast, and thatthe cell tolerates significant changes in NADP(H) reductionwithout deleterious effects.     

Down-regulation of trypsinogen expression is associated with growth retardation in alpha1,6-fucosyltransferase-deficient mice: attenuation of proteinase-activated receptor 2 activity

Alpha1,6-fucosyltransferase (Fut8) plays important roles inphysiological and pathological conditions. Fut8-deficient (Fut8^–/–)mice exhibit growth retardation, earlier postnatal death, andemphysema-like phenotype. To investigate the underlying molecularmechanism by which growth retardation occurs, we examined themRNA expression levels of Fut8^–/– embryos (18.5days postcoitum [dpc]) using a cDNA microarray. The DNA microarrayand real-time polymerase chain reaction (PCR) analysis showedthat a group of genes, including trypsinogens 4, 7, 8, 11, 16,and 20, were down-regulated in Fut8^–/– embryos.Consistently, the expression of trypsinogen proteins was foundto be lower in Fut8^–/– mice in the duodenum, smallintestine, and pancreas. Trypsin, an active form of trypsinogen,regulates cell growth through a G-protein-coupled receptor,the proteinase-activated receptor 2 (PAR-2). In a cell culturesystem, a Fut8 knockdown mouse pancreatic acinar cell carcinoma,TGP49-Fut8-KDs, showed decreased growth rate, similar to thatseen in Fut8^–/– mice, and the decreased growth ratewas rescued by the application of the PAR-2-activating peptide(SLIGRL-NH₂). Moreover, epidermal growth factor (EGF)-inducedreceptor phosphorylation was attenuated in TGP49-Fut8-KDs, whichwas highly associated with a reduction of trypsinogens mRNAlevels. The addition of exogenous EGF recovered c-fos, c-jun,and trypsinogen mRNA expression in TGP49-Fut8-KDs. Again, theEGF-induced up-regulation of c-fos and c-jun mRNA expressionwas significantly blocked by the protein kinase C (PKC) inhibitor.Our findings clearly demonstrate a relationship between Fut8and the regulation of EGF receptor (EGFR)-trypsin-PAR-2 pathwayin controlling cell growth and that the EGFR-trypsin-PAR-2 pathwayis suppressed in TGP49-Fut8-KDs as well as in Fut8^–/–mice.     

Studies on nucleic acids in plastids of the pigment mutant C-2A{acute} of Scenedesmus obliquus

Pigment mutant C-2A{acute} of Scenedesmus obliquus whose chlorophyllformation and chloroplast development are light dependent, wasstudied for the nucleic acid content of its plastids. The ribosomalRNA of plastids of the achlorophyllous or greened mutant C-2A{acute},did not show any difference from that of the wild type. Incorporationof [5-³H] uridine into mutant cells was partially inhibitedby rifampicin, indicating this part as being plastidial incorporation.Since there were no significant differences in the ribosomalRNA of plastids between the mutant and the wild type of Scenedesmus,the ribosomal system in the plastids of mutant C-2A' seemednot to be affected by the mutation. C_sCl gradient patterns ofScenedesmus mutant and wild-type DNA were almost identical withthose of Chlorella DNA. A peak at a buoyant density of 1.69g/cm³, the same as that of Chlorella chloroplast DNA, couldbe identified in Scenedesmus also as plastid DNA because itdisappeared after prolonged treatment with myxin and hybridizedwith rifampicin-sensitive pulse-labelled RNA. This peak waspresent to nearly the same degree in the mutant and the wildtype, indicating that a larger deficit of plastid DNA did notoccur in the mutant. Whether or not the mutation might be localizedin the plastid genome is discussed. (Received March 19, 1976; )     

Impermeability of the GIRK2 weaver channel to divalent cations

Asingle amino acid mutation (G156S) in the putative pore-forming regionof the G protein-sensitive, inwardly rectifying K⁺ channelsubunit, GIRK2, renders the conductance constitutively active andnonselective for monovalent cations. The mutant channel subunit(GIRK2wv) causes the pleiotropic weaver disease inmice, which is characterized by the selective vulnerability ofcerebellar granule cells and Purkinje cells, as well as dopaminergicneurons in the mesencephalon, to cell death. It has beenproposed that divalent cation permeability through constitutivelyactive GIRK2wv channels contributes to a rise in internalcalcium in the GIRK2wv-expressing neurons, eventually leadingto cell death. We carried out comparative studies of recombinantGIRK2wv channels expressed in Xenopus oocytes and COS-7cells to determine the magnitude and relative permeability of theGIRK2wv conductance to Ca²⁺. Data from thesestudies demonstrate that the properties of the expressed current differin the two systems and that when recombinant GIRK2wv isexpressed in mammalian cells it is impermeable to Ca²⁺.

     

Mutations that affect flightin expression in Drosophila alter the viscoelastic properties of flight muscle fibers

Striated muscles across phyla share a highly conserved sarcomere design yet exhibit broad diversity in contractile velocity, force, power output, and efficiency. Insect asynchronous flight muscles are characterized by high-frequency contraction, endurance, and high-power output. These muscles have evolved an enhanced delayed force response to stretch that is largely responsible for their enhanced oscillatory work and power production. In this study we investigated the contribution of flightin to oscillatory work using sinusoidal analysis of fibers from three flightless mutants affecting flightin expression: 1) fln⁰, a flightin null mutant, 2) Mhc¹³, a myosin rod point mutant with reduced levels of flightin, and 3) Mhc⁶, a second myosin rod point mutant with reduced levels of phosphorylated flightin. Fibers from the three mutants show deficits in their passive and dynamic viscoelastic properties that are commensurate with their effect on flightin expression and result in a significant loss of oscillatory work and power. Passive tension and passive stiffness were significantly reduced in fln⁰ and Mhc¹³ but not in Mhc⁶. The dynamic viscous modulus was significantly reduced in the three mutants, whereas the dynamic elastic modulus was reduced in fln⁰ and Mhc¹³ but not in Mhc⁶. Tension generation under isometric conditions was not impaired in fln⁰. However, when subjected to sinusoidal length perturbations, work-absorbing processes dominated over work-producing processes, resulting in no net positive work output. We propose that flightin is a major contributor to myofilament stiffness and a key determinant of the enhanced delayed force response to stretch in Drosophila flight muscles. flight muscles; muscle mutants; myosin     

A Plasma Membrane Mutation acrA in Escherichia coli

Protein analysis and electron microscopic observation of thefreeze-fractured plane of the plasma membrane were performedwith an acriflavine-sensitive mutant carrying mutation acrA(at min 10) and with the wild type (acrA⁺) strain of Escherichiacoli K-12. The acrA mutant membrane was deficient (or much lower)in one protein when analyzed by the polyacrylamide gel electrophoresistechnique. (Received May 7, 1981; Accepted July 28, 1981)     

Targeted inactivation of the gene psaK encoding a subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Mutant strains of the unicellular cyanobacterium Synechocystissp. PCC 6803, in which the psaK gene was insertionally inactivatedby targeted mutagenesis, were constructed. The gene is one ofthe two potential PsaK-coding genes which have been found asa result of the genome project with this cyanobacterium. Oneof the mutants was characterized in detail. A monocistronic,480-nucleotide mRNA of psaK was absent in total RNA from themutant cells. Inactivation of psaK had little effect on theaccumulation of polypeptides in the isolated PSI complexes exceptfor a polypeptide with an apparent molecular mass of 4.6 kDawhich was absent in the mutant. The amino-terminal amino acidsequence of the 4.6-kDa polypeptide confirmed that it was thetranslation product of psaK and further revealed a presequenceof PsaK. Characteristics of photoautotrophic growth at differenttemperatures, the amount of chlorophyll per cell, photosyntheticelectron transport rates with various electron acceptors, thekinetics of charge recombination between P700⁺ and reduced F_A/F_B,and the molar ratio of chlorophyll to P700, of the mutant werenot significantly different from those of the wild type. Furthermore,the trimer to monomer ratio of the PSI complexes isolated fromthe mutant was similar to that isolated from the wild type. (Received July 27, 1998; Accepted October 13, 1998)     

Uptake of 32P by Young Plants of Banksia hookeriana Meissner When Healthy and Infected with Phytophthora cinnamomi Rands

Young plants of Banksia hookeriana were grown in acid-washedsand with adequate phosphate and water supply, and a proportionwere inoculated with Phytophthora cinnamomi. There were no majordifferences in growth between uninoculated and infected plants,but there was a large increase in uptake of ³²P with increasingroot disease. In healthy plants ³²P uptake was greatest in youngleaf tissue, but in diseased plants labelled phosphate was directedmore towards older leaves where the activity was almost twicethat of young leaves. Enhanced uptake with disease was ascribed to possible blockageof the ‘message’ or ‘signal’ of phosphatetranslocation from shoot to root, such that the diseased rootincorrectly treated the shoot as P deficient and increased Puptake. Key words: Banksia hookeriana, Proteaceae, ³²P uptake, Phytophthora cinnamomi     

Photoinhibition and light-induced cyclic electron transport in ndhB(-) and psaE(-) mutants of Synechocystis sp. PCC 6803

The ndhB^– and psaE^– mutants of the cyanobacteriumSynechocystis sp. PCC 6803 are partly deficient in PSI-drivencyclic electron transport. We compared photoinhibition in thesemutants to the wild type to test the hypothesis that PSI cyclicelectron transport protects against photoinhibition. Photoinhibitorytreatment greatly accelerated PSI cyclic electron transportin the wild type and also in both the mutants. The psaE^–mutant showed rates of PSI cyclic electron transport similarto the wild type under all conditions tested. The ndhB^–mutant showed much lower rates of PSI cyclic electron transportthan the wild type following brief dark adaptation but exceededwild type rates after exposure to photoinhibitory light. Thewild type and both mutants showed similar rates of photoinhibitiondamage and photoinhibition repair at PSII. Photoinhibition atPSI was much slower than at PSII and was also similar betweenthe wild type and both mutants, despite the known instabilityof PSI in the psaE^– mutant. We conclude that photoinhibitorylight induces sufficient PSI-driven cyclic electron transportin both the ndhB^– and psaE^– mutants to fulfill anyrole that cyclic electron transport plays in protection againstphotoinhibition. ⁴ Corresponding author: E-mail, sherbert@uwyo.edu; Fax, +1-307-766-2851;Phone, +1-307-766-4353.     

Neurophysiological Genetics in Drosophila melanogaster

Neurophysiological genetics is the study of the mechanisms bywhich genes control nervous function and behavior. The transductionof genetic information into neural information is studied atthe level of the neuron through genetic and physiological techniques. The neurons responsible for the leg-shaking action specificto a single-gene mutant of Drosophila melanogaster, Hk¹, havebeen located in three pairs of small regions in the thoracicganglion. The activity pattern of these neurons is coded bythe mutant Hk¹ gene. The center for the specifically patternedleg-shaking action is composed of several motor neurons whoseactivity is governed by the pacemaking activity of at leastone interneuron. As it is most likely that the mutant gene isexpressed autonomously in this interneuron, there is a possibilityof investigating ways in which genes may influence the propertiesof neurons. The activity of the mutant neuron was monitoredintracellularly, and the pattern formation mechanism was studied.The amplitude, duration, and periodicity of the pacemaker potentialand the spike initiation site determine the activity patternresulting in the specific leg-shaking action.