Autonomy for a specific gene product in oocytes: Experimental evidence in the polychaetous annelid,Platynereis dumerilii

Oocytes of Platynereis dumerilii in early vitellogenesis were injected into female worms with oocytes of similar diameter. The donor oocytes were labeled by the or gene controlling eye pigmentation and, after some weeks of growth, were spawned together with the host oocytes. In most cases, a few donor progeny could be found among the offspring produced by the hosts. Donor progeny were examined with respect to an or gene-dependent maternal effect which normally causes wild-type eye color in homozygous ($\text{or}\text{or}$) larvae originating from the crossings of heterozygous ($\text{or}{}^{\mathrm{+}}\text{or}$) females and homozygous ($\text{or}\text{or}$) males. This maternal effect was absent from homozygous ($\text{or}\text{or}$) larvae derived from homozygous ($\text{or}\text{or}$) donor oocytes which had developed in heterozygous females. Conversely, this maternal effect was observed in homozygous ($\text{or}\text{or}$) larvae derived from heterozygous ($\text{or}{}^{\mathrm{+}}\text{or}$) donor oocytes which had developed in homozygous ($\text{or}\text{or}$) host females. It is concluded that the oocyte genome is active at the or⁺ locus during oogenesis and that the oocyte is autonomous with respect to the product of synthesis of the or⁺ locus. In the present case, the “maternal effect” is therefore caused by synthetic activity in the growing oocyte. The results are discussed with respect to current information on gene products from animal genomes.     

A mutant affecting the crystal cells inDrosophila melanogaster

Summary Black cells (Bc, 2-80.6±) mutant larvae ofDrosophila melanogaster have pigmented cells in the hemolymph and lymph glands. In this report we present evidence that these melanized cells are a mutant form of the crystal cells, a type of larval hemocyte with characteristic paracrystalline inclusions.Bc larvae lack crystal cells. Furthermore, the distribution pattern of black cells inBc larvae parallels that of experimentally-blackened crystal cells in normal larvae (phenocopy).InBc/Bc zygotes black cells appear during mid embryonic development but inBc ⁺/Bc zygotes pigmented cells are not found until late in the first larval instar.Crystal cells are present in the heterozygous larvae until this time, and paracrystalline inclusions can be seen in some of the cells undergoing melanization in these larvae.The rate of phenol oxidase activity inBc ⁺/Bc larval cell-free extracts is less than half that ofBc ⁺/Bc ⁺extracts whereas enzyme activity is undetectable inBc/Bc larvae. We propose that theBc ⁺gene product is required for maintaining the integrity of the paracrystalline inclusions; inBc/Bc larvae either the product is absent or nonfunctional so an effective contact between substrate and enzyme results in melanization of the cells.Phenol oxidase itself is either destroyed or consumed in the melanization process accounting for the absence of enzyme activity inBc/Bc larvae. These studies confirm that the crystal cells store phenolic substrates and are the source of the hemolymph phenol oxidase activity in the larva ofD. melanogaster.     

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     

Genetic control of DNA breakdown and repair inE. coli K-12 treated with mitomycin C or ultraviolet light

Summary Ultraviolet light sensitive mutants ofE. coli defective at theuvrA,uvrB oruvrC locus showed increased sensitivity to the lethal effects of mitomycin C when compared with theuvr ⁺ parental strain. In addition, DNA breakdown after treatment of cells with either mitomycin C or with ultraviolet light was greater in the parental strain carrying the activeuvr ⁺ genes than inuvr mutants. Thus, injuries produced by either mitomycin C or by ultraviolet light may be repaired by the same molecular mechanism which has been proposed and which involves defect excision, single strand breakdown and reconstruction of the DNA.With 2 Figures in the Text     

Interaction of alleles of therelA, relC andspoT genes inEscherichia coli: Analysis of the interconversion of GTP, ppGpp and pppGpp

Summary Mutants in thespoT gene have been isolated as stringent second site revertants of therelC mutation. These show varying degrees of the characteristics associated with thespoT1 gene,viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of³H-guanosine into GTP and ppGpp pools inspoT ⁺ andspoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower inspoT ^- than inspoT ⁺ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower inspoT than inspoT ⁺ cells. In one of the “intermediate”spoT mutants the rate of entry of³H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is:GDP→GTP→pppGpp→ppGpp→Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in variousspoT ⁺ andspoT ^- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis. ThespoT1 allele was introduced into various relaxed mutants. It was shown that many phenomena associated with the relaxed response ofrelC and “intermediate”relA mutants were phenotypically suppressed when thespoT1 allele was introduced into these mutants. These double mutants exhibit ppGpp accumulation, rate of RNA accumulation, rate of β-galactosidase synthesis, and heat lability of β-galactosidase synthesized during amino acid starvation similar to the stringent wild-type. It is concluded that the relaxed response is due directly to the lack of ppGpp and that the stringest response is due directly to ppGpp.     

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.     

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.     

Zur genphysiologischen Analyse der Pterine im Insektenauge (Drosophila melanogaster undCalliphora erythrocephala)

Summary The action of genew ^bl andw ^m4 on the eye-pteridines ofDrosophila melanogaster under the influence of different temperatures is studied. Whereas inw ^bl the temperature unfavorable for the synthesis of the eye-pteridines (25°C) results in a marked decrease of all pteridines, inw ^m4 the unfavorable temperature (18°C) results in a decrease of the red pteridine, but causes an accumulation of the tetrahydrobiopterin-compound and the yellow pteridine. InCalliphora erythrocephala genew causes accumulation of the yellow pteridine but a marked decrease of the tetrahydrobiopterin-compound. The relationships between the tetrahydrobiopterin-compound, the yellow pteridine (which probably is the dihydro-product) and the red colored end-product are discussed.

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Isolation of a new paramutagenic allele of thesulfurea locus in the tomato cultivar Moneymaker following in vitro culture

A new allele, SC148, of thesulfurea locus inLycopersicon esculentum was detected in a line derived after repeated selfing of plants that had been regenerated from tissue culture. Like the originalsulf mutant, SC148 displayed two mutant phenotypes: green-yellow speckled plants in which thesulf ^vag allele is present and pure yellow plants homozygous for thesulf ^tpura allele. Although the mutant alleles are recessive to wild-type, an unpredictable number of variegated and pura plants appeared in F₁ progenies that had been derived from crosses between SC148 and wild-type tomato plants. The presence of the wild-typesulf ⁺ allele in these variegated heterozygotes was demonstrated using a cytological marker that is linked tosulf. It is concluded that the mutantsulf allele of SC148, imposes its variegated expression state on the wild-typesulf ⁺ allele present insulf ⁺/sulf^vag heterozygotes. This behaviour, known as paramutation, has also been described for the originalsulf allele. The SC148 allele, however, seems to induce changes at an earlier stage in development. The analogy of this paramutagenic system to dominant position effect variegation inDrosophila is discussed.     

Genetic regulation of theAchaete-scute complex ofDrosophila melanogaster

Summary Mutants in two loci,hairy (h ⁺) andextramacrochaetae (emc ⁺), produce phenotypes corresponding to an excess of function of theachaete-scute complex (AS-C), that is, they cause the appearance of extra chaetae. These mutants, although recessive in normal flies, become dominant in the presence of extra doses of AS-C. Here we study the interactions between these three genes, in an attempt to elucidate their relationships. The results show that the insufficiency produced byh oremc mutants can be titrated by altering the number of copies of AS-C. Moreover, excess of function of AS-C produced by derepression mutants within the complex (Hairy-wing) can also be titrated by altering the number of wild type copies of⁺ oremc ⁺. These specific interactions indicate that bothh ⁺ andemc ⁺ code for repressors of AS-C that interact with theachaete andscute region of the complex respectively.     

Relationship between the F0F1-ATPase and the K+-transport system within the membrane of anaerobically grownEscherichia coli. N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity in mutants with defects in K+-transport

A considerable (2-fold) stimulation of the DCCD-sensitive ATPase activity by K⁺ or Rb⁺, but not by Na⁺, over the range of zero to 100mM was shown in the isolated membranes ofE. coli grown anaerobically in the presence of glucose. This effect was observed only in parent and in thetrkG, but not in thetrkA, trkE, ortrkH mutants. ThetrkG or thetrkH mutant with anunc deletion had a residual ATPase activity not sensitive to DCCD. A stimulation of the DCCD-sensitive ATPase activity by K⁺ was absent in the membranes from bacteria grown anaerobically in the presence of sodium nitrate. Growth of thetrkG, but not of othertrk mutants, in the medium with moderate K⁺ activity did not depend on K⁺ concentration. Under upshock, K⁺ accumulation was essentially higher in thetrkG mutant than in the othertrk mutant. The K⁺-stimulated DCCD-sensitive ATPase activity in the membranes isolated from anaerobically grownE. coli has been shown to depend absolutely on both the F₀F₁ and theTrk system and can be explained by a direct interaction between these transport systems within the membrane of anaerobically grown bacteria with the formation of a single supercomplex functioning as a H⁺-K⁺ pump. ThetrkG gene is most probably not functional in anaerobically grown bacteria.This study was performed at the Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637.     

Mutations inmotBSuppressible by Changes in Stator or Rotor Components of the Bacterial Flagellar Motor

Five proteins (MotA, MotB, FliG, FliM and FliN) may be involved in energizing flagellar rotation inEscherichia coli. To study interactions between the Mot proteins, and between them and the three Fli proteins of the switch-motor complex, we have isolated extragenic suppressors of dominant and partially dominantmotBmissense mutations. Four of the 13motBmutations yielded partially allele-specific suppressors. Of the suppressing mutations, 57 are in themotAgene, eight are infliG, and one is infliM; no suppressor was identified infliN. The prevalence of suppressors infliGsuggests that FliG interacts rather directly with the Mot proteins. The behaviour of cells in tethering and swarm assays indicates that themotAsuppressors are more efficient than thefliGorfliMsuppressors. Some of the suppressing mutations themselves confer distinctive phenotypes inmotB⁺cells. We propose a model in which mutations affecting residues in or near the putative peptidoglucan-binding region of MotB misalign the stator relative to the rotor. We suggest that most of the suppressors restore motility by introducing compensatory realignments in MotA or FliG.     

Comparison of a lipophilic cation and microelectrodes to measure membrane potentials of the giant-celled algae,Chara australis (Charophyta) andGriffithsia monilis (Rhodophyta)

Summary The vacuolar equilibrium potential of the lipophilic cation TPMP⁺ (triphenyl methyl phosphonium) in the giant algaeChara australis andGriffithsia monilis was directly measured. The TPMP⁺ equilibrium potential was approximately 100mV less negative than the measured vacuolar electrical potential. Thus TPMP⁺ does not act as a probe of the vacuolar electrical potential and appears to be extruded against an electrochemical gradient. Measurement of the plasmalemma equilibrium potential of TPMP⁺ showed that extrusion of TPMP⁺ apparently occurred at both the tonoplast and plasmalemma inChara and at the plasmalemma inGriffithsia. It is concluded that TPMP⁺ cannot be used as a membrane potential probe inChara orGriffithsia.     

Reduced transmitter release conferred by mutations in theslowpoke-encoded Ca2+-activated K+ channel gene ofDrosophila

Potassium channels control the repolarization of nerve terminals and thus play important roles in the control of synaptic transmission. Here we describe the effects of mutations in theslowpoke gene, which is the structural gene for a calcium activated potassium channel, on transmitter release at the neuromuscular junction inDrosophila melanogaster. Surprisingly, we find that theslowpoke mutant exhibits reduced transmitter release compared to normal. Similarly, theslowpoke mutation significantly suppresses the increased transmitter release conferred either by a mutation inShaker or by application of 4-aminopyridine, which blocks theShaker-encoded potassium channel at theDrosophila nerve terminal. Furthermore, theslowpoke mutation suppresses the striking increase in transmitter release that occurs following application of 4-aminopyridine to theether a go-go mutant. This suppression is most likely the result of a reduction of Ca²⁺ influx into the nerve terminal in theslowpoke mutant. We hypothesize that the effects of theslowpoke mutation are indirect, perhaps resulting from increased Ca²⁺ channel inactivation, decreased Na⁺ or Ca²⁺ channel localization or gene expression, or by increases in the expression or activity of potassium channels distinct fromslowpoke.     

Modulation of gene expression by the product offitA gene inEscherichia coli

Physiological parameters such as viability, gross RNA synthesis,β-galactosidase induction, development of phages T4, T7 andλ have been studied in temperature-sensitiveEscherichia coli strains harbouring fit A76,fit A24 andfit A76fit A24 mutations in rpoB⁺ andrpoB240 genetic backgrounds. The efficiently of expression of these functions is influenced by thefit A alleles depending upon the medium of growth and/or temperature. Strains harbouring therpoB240 mutation and thefit A76 mutation, either alone or together with thefit A24 mutation, are rifampicin-sensitive even at the perfssive temperature. The results suggest possible interaction between thefit A gene product and RNA polymerase invivo. This paper is dedicated to Proof. S. Krishnaswamy on his Sixty First Birthday.     

The maternal and zygotic control of development by cinnamon, a new mutant in Drosophila melanogaster

A genetic analysis of the X-linked mutant cinnamon (cin) in Drosophila melanogaster demonstrates that this locus controls a process that is essential for zygote survival. Survival of a zygote is shown to be ensured by either the maternal expression of the cin⁺ allele or by the presence of a cin⁺ allele in the zygotic genome. The cin locus is also shown to affect pteridine pigment metabolism. The presence of a cin⁺ allele in either the mother or zygote is requisite for normal zygotic eye pigmentation. It is shown that cin flies lack xanthine dehydrogenase, an enzyme involved in pteridine metabolism, and accumulate the enzyme's pteridine and purine substrates. The relationship between the effects of cin on viability and xanthine dehydrogenase activity is discussed.     

Importance ofeps genes fromBacillus subtilis in biofilm formation and swarming

Unicellular organisms naturally form multicellular communities, differentiate into specialized cells, and synchronize their behaviour under certain conditions. Swarming, defined as a movement of a large mass of bacteria on solid surfaces, is recognized as a preliminary step in the formation of biofilms. The main aim of this work was to study the role of a group of genes involved in exopolysaccharide biosynthesis during pellicle formation and swarming inBacillus subtilis strain 168. To assess the role of particular proteins encoded by the group ofepsI-epsO genes that form theeps operon, we constructed a series of insertional mutants. The results obtained showed that mutations inepsJ-epsN, but not in the last gene of theeps operon (epsO), have a severe effect on pellicle formation under all tested conditions. Moreover, the inactivation of 5 out of the 6 genes analysed caused total inhibition of swarming in strain 168 (that does not produce surfactin) on LB medium. Following restoration of thesfp gene (required for production of surfactin, which is essential for swarming of the wild-type bacteria), thesfp ⁺ strains defective ineps genes (exceptepsO) generated significantly different patterns during swarming on synthetic B medium, as compared to the parental strain 168sfp ⁺.     

OriVRK2 replicon function in the absence oftrfA inAzotobacter vinelandii

The oriV_RK2 does not need the function of either trfA⁺ ortrfA Operon for replication and maintenance of an oriV_RK2-containing plasmid inAzotobacter vinelandii.     

High-level expression of the colicin A lysis protein

Summary Two plasmids that overproduce the colicin A lysis protein, Cal, are described. Plasmid AT1 was constructed by a deletion in the colicin A operon, which placed thecal gene near a truncatedcaa gene in such a way that both gene products were synthesized at high levels following induction. Plasmid Ck4 was constructed by insertion of thecal gene downstream from thetac promoter of an expression vector. Overproduction of Cal was obtained after mitomycin C induction of pAT1 cells and after IPTG induction of pCK4 cells. The kinetics of Cal synthesis were examined with [³⁵S] methionine and [2-³H] glycerol inlpp orlpp ⁺ host strains. Each of the steps of the lipid modification and maturation pathway of Cal was demonstrated. The modified precursor form of overproduced Cal was not chased as efficiently as when it is produced in pColA cells. After treatment with globomycin, a significant amount of this modified precursor form accumulated and was degraded with time into smaller acylated proteins, but without release of the signal peptide. Release of cellular proteins and quasi-lysis were observed after about 1 hour of induction for cells containing either plasmid. In addition, in Cal-overproducing cells, the rate of quasi-lysis was increased but not its extent. InpldA cells, quasi-lysis was reduced but not abolished. Lethality of the Cal induction in the overproducing cells was in the same range as that in wild-type cells.     

TheUbx syndrome ofDrosophila: the prothoracic transformation (ppx) is independent ofbx,bxd andpbx

Summary If, early in development, theUbx ⁺ gene is removed by mitotic recombination from cells of the meso-and metathoracic leg primordia, theseUbx ^– cells develop as in the posterior prothoracic leg. We show that this transformation, termedpostprothorax, is a discrete genetic function that is independent of other homeotic transformations such asbx, pbx orbxd, which also result from the inactivation of theUbx gene.