Cell interactions in the generation of chaetae pattern inDrosophila

Summary We have already shown that theachaetae-scute complex (AS-C) ofDrosophila is regulated by two genes,hairy andextramacrochaetae. Using mutants in these genes, we have analysed how different levels of expression of AS-C affect the pattern of chaetae. The results indicate that the spatial distribution of chaetae results from cell interactions, probably by a mechanism of lateral inhibition. The results are discussed in view of the different theories of pattern formation.     

Genetic analysis ofHairy-wing mutations

Summary We studied the genetic bases of threeHairy-wing (Hw ¹,Hw ^Ua ,Hw ^49c ) mutations mapping in the region of theachaete-scute complex (AS-C). Analysis of X-ray-induced revertants ofHw ¹ andHw ^49c uncoveredachaete andscute mutant phenotypes respectively. This indicates that theHw mutant phenotypes result from an excess of function of these genes of theachaete-scute complex (AS-C). The phenotypes of the differentHws show allelic specificity in the pattern of extrachaetes. In addition to these mutations, certain inversions and internal duplications of the AS-C also produce aHw-variegated phenotype, probably due to variegation or decompensation of the genes of the AS-C. The expressivity of the differentHws (mutation or variegation) is modulated by the number of doses of the AS-C present in the genome. A similar dose-dependent modulation is exerted by the transregulatory geneshairy andextramacrochaetae. We discuss these results on the basis of a regulation model of the expression of the AS-C.     

Contribution of the geneextramacrochaetae to the precise positioning of bristles inDrosophila

We examine the effect of mutations in theextramacrochaetae (emc) gene on the positioning of macrochaetes on the notum ofDrosophila. We show that, inemc mutants, most of the precursor cells appear earlier than in wild-type individuals, consistent with an antagonizing effect ofemc on the action of the proneural genesachaete andscute. We also show that reducingemc function affects the position of three bristles and/or of their precursors, but has no marked effect on the positioning of the other bristles.     

Transfer ofF′ fromEscherichia coli K 12 toEscherichia coli B and to strains ofParacolobacter andKlebsiella

The transfer of theF episome fromEscherichia coli K 12 toE. coli B,Paracolobacter andKlebsiella was studied. The frequency of transfer of the episomal markers toE. coli B was very low. The large majority ofE. coli B cells which had received the episomal markerslac ⁺ orgal ⁺ were F^–, which indicates that the episomal markers were stably integrated on the chromosome. Recombinants from K 12 F⁺ × B F^– crosses were mostly F^–. These results suggest that the multiplication of theF-factor ofE. coli K 12 is restricted inE. coli B. The transfer of theF-lac ⁺ Ad ⁺ episome fromE. coli K 12 toParacolobacter andKlebsiella strains was in most cases only possible when donor and acceptor strain were plated together on selective media. Stable incorporation of episomal markers was also found withParacolobacter coliforme. Paracolobacter aerogenoides andKlebsiella aerogenes strains could be infected withF-lac ⁺ Ad ⁺. The episomal markers were not incorporated and the episomes were easily lost, which indicates that these strains contained theF factor in the autonomous state.     

The effect of spo0 mutations on the expression of spo0A- and spo0F-lacZ fusions

Summary We have constructedspo0A-lacZ andspo0F-lacZ fusions with a temperate phage vector and have investigated howspo0 gene products are involved in the expression of each of these genes. The expression ofspo0A-lacZ andspo0F-lacZ was stimulated at about the time of cessation of vegetative growth in Spo⁺ cells. This stimulation ofspo0A-lacZ was impaired by mutations in thespo0B, D, E, F orH genes but was not affected by mutations in thespo0J orK genes. Similar results were obtained with thespo0F-lacZ fusion. The effect of thespo0A mutation onspo0A-lacZ expression was characteristic: thespo0A-directed β-galactosidase activity found during vegetative growth was significantly enhanced in thespo0A mutant. This result suggests thatspo0A gene expression is autoregulated being repressed by its own gene product. Another remarkable observation was the effect of thesof-1 mutation, which is known to be aspo0A allele; it suppressed the sporulation deficiency ofspo0B, spo0D andspo0F mutants. Thespo0A-lacZ stimulation, which is impaired by any one of thesespo0 mutations, was restored by the additionalsof-1 mutation.     

Functional characterisation of two cytochrome<Emphasis Type="Italic"> b</Emphasis><Subscript>5</Subscript>-fusion desaturases from<Emphasis Type="Italic"> Anemone leveillei</Emphasis>: the unexpected identification of a fatty acid Δ<Superscript>6</Superscript>-desaturase

The Ranunculaceae are known to accumulate a wide range of unusual fatty acids in their seed lipids, and this variability has been advocated as a taxonomic marker. The Anemone species, Anemone leveillei L. and Anemone rivularis Buch.-Ham., have previously been reported to accumulate ⁵-desaturated fatty acids in their seed tissue [K. Aitzetmüller (1995) Plant Syst Evol 9:229–240]. Two cDNAs, AL1 and AL2, with similarity to plant cytochrome b₅-fusion "front-end" desaturases were isolated from developing seeds of A. leveillei and their function identified by expression in Saccharomyces cerevisiae. AL2 was characterised as a sphingolipid long-chain-base ⁸-desaturase, while AL1 acted as a fatty acid desaturase. However, AL1 did not produce ⁵-desaturated fatty acids as expected; instead, when expressed in transgenic S. cerevisiae or Arabidopsis thaliana this enzyme was functionally characterised as a ⁶-desaturase. Northern analysis confirmed the expression of this gene in seed tissue and leaf tissue of A. leveillei, though ⁶-desaturated fatty acids were found to accumulate only in the leaf tissue. The unexpected characterisation of a ⁶-desaturase in A. leveillei has implications for the use of fatty acids in chemotaxonomic studies. This is also the first report of a higher-plant ⁶-desaturase from a family other than the Boraginaceae.Abbreviations ALA -linolenic acid - DMOX 4,4-dimethyloxazoline - EDA eicosadienoic acid - FAME fatty acid methyl ester - GLA -linolenic acid - LA linoleic acid - LCB long chain base - ORF open reading frame - OTA octadecatetraenoic acid     

Chromosomal location of a gene (idg) that specifies production of the blue pigment indigoidine inErwinia chrysanthemi

Production of a blue pigment, indigoidine, is a variable trait among wild-type strains ofErwinia chrysanthemi; it is also influenced by the composition of the growth medium. Starting with a nonpigmented wild-type strain (ICPB EC183) ofE. chrysanthemi, we obtained by nitrosoguanidine mutagenesis a pigmented (idg ⁺) mutant strain (AC6055), which simultaneously was Arg^–. Linkage betweenarg andidg was established in two-factor transductional and conjugational crosses. Coinheritance ofidg withhis ⁺,ilv ⁺,leu ⁺,ser ⁺,thr ⁺, orura ⁺ transductants was not observed. Spontaneous Arg⁺ revertants of AC6055 were invariably Idg⁺. The pigments produced by AC6055, Idg⁺ recombinants, and wild-type Idg⁺ strains were identical, judged by absorption spectra (_max = 615 nm) of the dimethylsulfoxide extracts of whole cells. We concluded that nitrosoguanidine caused comutation in thearg andidg loci that are linked on theE. chrysanthemi chromosome.     

Mutations that are synthetically lethal with a<Emphasis Type="Italic"> gas1Δ</Emphasis> allele cause defects in the cell wall of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The GAS1 -related genes of fungi encode GPI-anchored proteins with -1,3-glucanosyltransferase activity. Loss of this activity results in defects in the assembly of the cell wall. We isolated mutants that show a synthetic defect when combined with a gas1 allele in Saccharomyces cerevisiae, and identified nine wild-type genes that rescue this defect. The indispensability of BIG1 and KRE6 for the viability of gas1 cells confirmed the important role of -1,6-glucan in cells that are defective in the processing of -1,3-glucan. The identification of the Wsc1p hypo-osmotic stress sensor and components of the PKC signal transduction pathway in our screen also confirmed that the cell wall integrity response attenuates the otherwise lethal gas1 defect. Unexpectedly, we found that the KEX2 gene is also required for the viability of the gas1 mutant. Kex2p is a Golgi/endosome-membrane-anchored protease that processes secretory preproteins. A cell wall defect was also found in the kex2 mutant, which was suppressible by multiple copies of the MKC7 or YAP3 gene, both of which encode other GPI-anchored proteases. Therefore, normal cell wall assembly requires proteolytic processing of secretory preproteins. Furthermore, the genes CSG2 and IPT1 were found to be required for normal growth of gas1 cells in the presence of 1 M sorbitol. This finding suggests that complex sphingolipids play a role in the hyper-osmotic response.Communicated by C. P. Hollenberg     

Phenocopies ofBithorax mutants

Summary Exposure to ether of wild-type embryos of different strains ofDrosophila melanogaster causes phenocopies of different alleles of thebithorax system. Clonal analysis of the phenocopy spots has shown that the transformation caused by the treatment is maintained by cell heredity. Embryos heterozygous for several recessive mutant alleles ofbithorax show the same frequency of phenocopies as wild-type homozygous sib controls. The same holds for embryos heterozygous for the dominant mutant allelesCbx andUbx ¹ which are point mutants in thecis-regulatory region of the system. However, for dominant mutants which have breakpoints in this region (Ubx ⁸⁰,Ubx ¹³⁰ andHm) the frequency of phenocopies is about twice that of their sib controls. Embryos with increasing numbers of copies (from 1 to 4) of thebithorax system show a decreasing frequency of phenocopies. A model is proposed that explains bithorax phenocopies as resulting from disturbances in the distribution of positional information signals for segments (inductor molecules) which compete with the product of a regulator gene (repressor) and thecis-regulatory region of thebithorax system. On this model, the initiation of a metathoracic developmental pathway would result from the derepression of thebithorax system.     

<Emphasis Type="Italic"> Cis</Emphasis>-acting elements sufficient for induction of<Emphasis Type="Italic"> FDH1</Emphasis> expression by formate in the methylotrophic yeast<Emphasis Type="Italic"> Candida boidinii</Emphasis>

The FDH1 gene of Candida boidinii encodes an NAD⁺-dependent formate dehydrogenase, which catalyzes the last reaction in the methanol dissimilation pathway. FDH1 expression is strongly induced by methanol, as are the promoters of the genes AOD1 (alcohol oxidase) and DAS1 (dihydroxyacetone synthase). FDH1 expression can be induced by formate when cells are grown on a medium containing glucose as a carbon source, whereas expression of AOD1 and DAS1 is completely repressed in the presence of glucose. Using deletion analyses, we identified two cis-acting regulatory elements, termed UAS-FM and UAS-M, respectively, in the 5 non-coding region of the FDH1 gene. Both elements were necessary for full induction of the FDH1 promoter by methanol, while only the UAS-FM element was required for full induction by formate. Irrespective of whether induction was achieved with methanol or formate, the UAS-FM element enhanced the level of induction of the FDH1 promoter in a manner dependent on the number of copies, but independent of their orientation, and also converted the ACT1 promoter from a constitutive into an inducible element. Our results not only provide a powerful promoter for heterologous gene expression, but also yield insights into the mechanism of regulation of FDH1 expression at the molecular level.Communicated by C. P. Hollenberg     

The influence of pH and external H+ concentration on caesium toxicity and accumulation inEscherichia coli andBacillus subtilis

Summary Toxicity screening ofEscherichia coli NCIB 9484 andBacillus subtilis 007, NCIB 168 and NCIB 1650 has shown Cs⁺ to be the most toxic Group 1 metal cation. However, toxicity and accumulation of Cs⁺ by the bacteria was affected by two main external factors; pH and the presence of other monovalent cations, particularly K⁺. Over the pH range 6–9 bothE. coli andB. subtilis showed increasing sensitivity towards caesium as the pH was raised. The presence of K⁺ and Na⁺ in the laboratory media used lowered caesium toxicity and lowered acumulation of the metal. In order to assess accurately Cs⁺ toxicity towards the bacterial strains it was therefore necessary to define the K⁺:Cs⁺ ratio in the external medium. The minimum inhibitory K⁺:Cs⁺ concentration ratio for theBacillus strains tested was in the range 12–13 whileE. coli had a minimum inhibitory K⁺:Cs⁺ concentration ratio of 16.     

Characterization of<Emphasis Type="Italic"> Schizosaccharomyces pombe</Emphasis> mutants defective in vacuolar acidification and protein sorting

The vacuolar H⁺-ATPases (V-ATPases) are ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. To investigate the functional roles of the V-ATPase in Schizosaccharomyces pombe, the gene vma1 encoding subunit A or vma3 encoding subunit c was disrupted. Both deletion mutants lost the capacity for vacuolar acidification in vivo, and showed sensitivity to neutral pH or high concentrations of divalent cations including Ca²⁺. The delivery of FM4-64 to the vacuolar membrane and accumulation of Lucifer Yellow CH were strongly inhibited in the vma1 and vma3 mutants. Moreover, deletion of the S. pombe vma1 ⁺ or vma3 ⁺ gene resulted in pleiotropic phenotypes consistent with lack of vacuolar acidification, including the missorting of vacuolar carboxypeptidase Y, abnormal vacuole morphology, and mating defects. These findings suggest that V-ATPase is essential for endocytosis, ion and pH homeostasis, and for intracellular targeting of vacuolar proteins and vacuolar biogenesis in S. pombe.Communicated by M. Johnston     

Red cell H-deficient,salivary ABH secretor phenotype of Reunion island. Genetic control of the expression of H antigen in the skin

Based on the genetic model proposing thatH andSe are two structural genes, we predicted that the red cell H-deficient, salivary ABH secretor phenotype should be found on Reunion island, where a large series of H-deficient non-secretor families have been previously described. Two such Reunion individuals are now reported. POU [A_h, Le(a–b+), secretor of A, H, Le^a and Le^b in saliva] and SOU [O_h, Le(a–b+), secretor of H, Le^a and Le^b in saliva]. Both are devoid of H -2-fucosyltransferase activity in serum. In addition, the preparation of total non-acid glycosphingolipids from plasma and red cells of POU revealed the type 1ALe^b heptaglycosylceramide and small amounts of the monofucosylated type 1 A hexaglycosylceramide. Both glycolipids possess an H structure probably synthesised by the product of theSe gene. No other blood group A glycolipids, with types 2, 3 or 4 chains, normally present in the presence of the product of theH gene, were found on red cells or plasma of POU.TheH,Se andLe genetic control of the expression of ABH and related antigens in different tissue structures of the skin is described in 54 H-normal individuals of known ABO, secretor and Lewis phenotypes; in one red cell H-deficient salivary secretor (SOU); and in one H-deficient non-secretor (FRA). Sweat glands express ABH under the control of theSe gene. Sweat ducts express ABH under the control of bothH andSe genes and Lewis antigens under the control ofLe and bothH andSe genes. Epidermis, vascular endothelium and red cells express ABH under the control of theH gene. The products ofH andSe genes are usually expressed in different cells. However, the results illustrate that in some structures, like the epithelial cells of sweat ducts, both the products ofH andSe genes can contribute to the synthesis of the same Le^b structure.     

A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis inEscherichia coli

Different approaches to increasing carbon commitment to aromatic amino acid biosynthesis were compared in isogenic strains ofEscherichia coli. In a strain having a wild-type PEP: glucose phosphotransferase (PTS) system, inactivation of the genes encoding pyruvate kinase (pykA andpykF) resulted in a 3.4-fold increase in carbon flow to aromatic biosynthesis. In a strain already having increased carbon flow to aromatics by virtue of overexpression of thetktA gene (encoding transketolase), thepykA and/orpykF mutations had no effect. A PTS glucose⁺ mutant showed a 1.6-fold increase in carbon flow to aromatics compared to the PTS⁺ control strain. In the PTS^– glucose⁺ host background, overexpression oftktA caused a further 3.7-fold increase in carbon flow, while inactivation ofpykA andpykF caused a 5.8-fold increase. When all of the variables tested (PTS^– glucose⁺,pykA, pykF, and overexpressedtktA) were combined in a single strain, a 19.9-fold increase in carbon commitment to aromatic biosynthesis was achieved.     

Electron transport-linked proton translocation at nitrite reduction inCampylobacter sputorum subspeciesbubulus

Campylobacter sputorum subspeciesbubulus contains a membrane-bound nitrite reductase which catalyses the six-electron reduction of nitrite to ammonia. Formate andL-lactate are used as hydrogen donors. Cells ofC. sputorum grown with nitrate or nitrite contain cytochromes of theb-andc-type and a carbon monoxide-binding cytochromec. In addition, a special membrane-bound carbon monoxide-binding pigment is found. Nitrite reduction with formate orL-lactate as a hydrogen donor is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Nitrite reduction by bacterial suspensions with lactate as a hydrogen donor is strongly inhibited by carbonylcyanide-m-chlorophenyl-hydrazone (CCCP) whereas nitrite reduction with formate as a hydrogen donor is not inhibited at all. H⁺/O values and H⁺/NO ₂ ^- values were measured with ascorbate + N,N,N,N-tetramethyl-p-phenylenediamine (TMPD), formate (in the absence and presence of carbonic anhydrase) andL-lactate as a hydrogen donor. The results are summarized in a scheme for electron transport from formate or lactate to oxygen or nitrite which shows a periplasmic orientation of formate dehydrogenase and nitrite reductase and a cytoplasmic orientation of lactate dehydrogenase and oxygen reduction, and which shows proton translocation with a H⁺/2e value of 2.0. The H⁺/O and H⁺/NO ₂ ^- values predicted by this scheme are in good agreement with the experimental values.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - MTPP⁺ methyltriphenylphosphonium cation - TMPD N,N,N,N-tetramethyl-p-phenylenediamine; H⁺/O (H⁺/NO ₂ ^- ), number of protons liberated in the outer bulk phase at the reduction of one atom O (one ion NO ₂ ^- ); H⁺/2e (q⁺/2e), number of protons (charges) translocated across the cytoplasmic membrane during flow of two electrons to an acceptor     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

A mutation that increases a novel calcium-activated potassium conductance ofParamecium tetraurelia

Summary Under two-electrode voltage clamp, a mutant ofP. tetraurelia, restless (rst/rst), showed a large increase in induced current and an outward tail current when compared to the wildtype cell for hyperpolarizing voltage steps. An increase in the induced and tail currents is also observed for depolarizing voltage steps. The larger current during voltage steps and tail in the mutant were eliminated by the use of CsCl-filled electrodes and tetraethylammonium ion (TEA⁺) in the bath solution, characterizing the lesion as affecting a K⁺ conductance. Ionophoretic injection of ethylene glycol bis-(beta-aminoethyl ether) n,n,n,n-tetraacetic acid (EGTA) to buffer internal Ca²⁺ concentration reduced the increased K⁺ current and tail of therestless cell, indicating Ca²⁺ activation of the K⁺ current. Time course and amplitude of remaining currents after blockage of K⁺ conductances with Cs⁺ and TEA⁺ were similar in wild-type andrestless cells suggesting norestless defect in entry of calcium. The Ca²⁺-activated sodium current was similar in the mutant to that in wild type arguing against a defect in calcium regulation activating the K⁺ channel in therestless cell. We conclude that therestless mutation alters a Ca²⁺-activated potassium conductance other than the one previously described. The multiplicity of Ca²⁺-activated potassium conductances inParamecium is discussed.     

Improvement of growth and camptothecin yield by altering nitrogen source supply in cell suspension cultures of Camptotheca acuminata

Nitrate at 70m gave the highest biomass of Camptotheca acuminata in suspension culture in MS medium, but a NH₄ ⁺/NO₃ ^– molar ratio of 5:1 (giving a total of 40 m N) gave the maximum camptothecin yield. A two-stage flask culture system was established to improve culture efficiency; cell dry weight, camptothecin content and yield was increased by 30%, 280% and 340%, respectively when compared with those of control, reaching up to 36g l^–1, 0.36mgg^–1, and 12.8mgl^–1, respectively.     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium