A. Zaritsky

[CANCERRESEARCH30,498—503,February1970]SUMMARYTheelectrokineticsurfacepropertiesofchickembryocellculturestransformed byRoussarcomavirushavebeenstudiedforanyalterationsassociatedwiththemalignanttransformation. Inthissystemarapidandefficienttransformation invitrotakesplacewhichenablesacomparisontobemadebetweennormalandmalignantcellsderivedfromthesameembryoandculturedunderthesameconditions.NodifferencecouldbedetectedbetweenthenormalandmalignantcellsforeitherthesurfacechargeorpH-mobilityrelationship. Theexpecteddecreaseincalcium-bindingpowerofthemalignantcellswasnotevidencedinthissystem,asbothtypesofcellsalsohadthesameaffinitytocalcium.Thesedataaswellasotherrecentstudiessuggestthattherelationshipbetweencalciumaffinityandmalignancyisinneedoffurtherclarification.Neuraminidasetreatmentproducedamobilitydecreaseinmalignantcellsthatneversurpassedthatnotedinnormalcellsbutwasalwayseitherequivalenttoorlessthanthatoftheirnormalcounterparts.Itwasconcludedthatnogeneralization could bemadeastothecorrelation betweenmalignancy,increasedsurfacechargedensity,andincreasedsurfacesialicacid.INTRODUCTIONInpreviousstudiesattemptshavebeenmadetocorrelatechangesinsurfacepropertiesofthemalignantcellswiththeiruniquebiologicalbehavior.Ithasbeensuggestedthatthelackof“contactinhibitionâ€softhetumorcellswasrelatedtotheirpoorermutualadhesiveness(1)andwasalsoevidencedinadecreasedcalciumaffinityoftheirsurfaces(7).Insomecases(1,14,25)themalignantpropertieswerebelievedtobeassociatedwithahighernetsurfacenegativechargeduetoanincreasedsurfacesialicacid(3,14,26).Conflictingresultsfromtheliteratureastotherelationshipbetweenincreasedsurfacechargedensityandincreasedsurfacesialicacidofneoplasticcells(9,28,30)haveemphasizedtheneedforfurtherinvestigation ofthesesuppositions.Inthepresentstudyanattempthasbeenmadetoexaminetheaboveproblemsinawell-controlledsystemofneoplastictransformation invitro.TheRoussarcomavirusinducesarapidandalmostcompletetransformationinchickembryocellculturesinthecourseofafewdays(29).Incontrastotheroncogenicvirusestransformonlyasmallfractionofthecellsinculture,thusnecessitatinglong-termserialpassaging.Inthisinvestigation anelectrokinetic characterizationofnormalandRSV2-transformedchickembryocellshasbeenundertakentotestthevalidityoftheaboveassumptions.SpecialattentionhasbeengiventotheeffectsofneuraniinidasetreatmentandchangingpHandcalciumconcentrationontheelectrophoreticmobiitiesofthesecells.MATERIALSANDMETHODSCells.Primarycultureswerepreparedfrom9-to10-day-oldchickembryos.EggswereobtainedfrombrownLeghornchickens ofaleukosis-free flock(obtained throughthecourtesy ofDr.A.Kohn theIsraelInstituteforBiologicalResearch).Virus.TheBryan“high-titerâ€sstrainRSV(RAV-2)usedherewasobtainedfromDr.A.Kohn.Theviralstockwaspreparedfroma9-day-oldtumorproducedbywing-webinoculation.Media.Thestandard culturemediacontained M-199-5%calfserum-10%Tryptosephosphatewiththeusualconcentrationofpenicillin,streptomycin,andmycostatin.Solutions.Thestandardsolutionusedforelectrophoreticmeasurement was0.0145MNaCl,madeisotonicbytheadditionof4.8%glucoseandbufferedtoneutralitywith03

In an attempt to endow Cyt1Ca with Cyt1Aa-like antibacterial activity, both derived from Bacillus thuringiensis subsp. israelensis, two amino acids were replaced, E117V and N125A, so as to raise the hydrophobicity of the corresponding region, considered to be the membrane-active motif. The clones obtained included multiple repeats of VIEVLKSLLGIALA, corresponding to head-to-tail polymerization of the primer, translated in frame with Cyt1Ca. These versions of Cyt1Ca caused instant arrest in biomass growth and decreased viability upon expression in Escherichia coli. Multiple insertions of the non-mutated motif VIEELKSLLGINLA into the polypeptide were also lethal. To expose toxicity of the latter motif in the original Cyt1Ca, cyt1Ca was appropriately truncated.

The P19 gene and cyt1A gene were obtained by PCR with 9.7 kb HindIII fragment containing P19 gene and cyt1A gene from 72 MD plasmid of Bt as a template. Digested PCR products were ligated to expression vector pUHE24 and transformed into E. coli XL-1. Three clones, LZ19 harboring P19 gene, LZcyt1A harboring cyt1A gene, LZ19A harboring P19 gene and cyt1A gene, were screened. The growth cure of cloned strains were determined under IPTG induction. The result showed that pLZ19 did not affect the growth of E. coli, pLZcyt1A is typically Lethal for E. coli, Lethal initial efficiency of pLZ19A is much higher than that of pLZcyt1A. Probably, this was a result that P19 gene enhanced initial expression of cyt1A gene in E. coli.

— Division planes in Escherichia coli, usually restricted to one dimension of the rod-shaped cell, were induced at all possible planes by transforming the cells to spheroids with mecillinam (inactivating PbpA). Such cells displayed many nucleoids and arcs of FtsZ, genetically tagged to green fluorescent protein, that developed to rings at constriction sites all around their surface. These observations are consistent with the view (Woldringh et al., J. Bacteriol. 176 (1994) 6030-6038) that nucleoids, forced during replication to segregate in the length axis of the cell by the rigid bacillary envelope, induce assembly of FtsZ to division rings in between them. © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS

Various subspecies (ssp.) of Bacillus thuringiensis (Bt) are considered the best agents known so far to control insects, being highly specific and safe, easily mass produced and with long shelf life.1 The para-crystalline body that is produced during sporulation in the exosporium includes polypeptides named δ-endotoxins, each killing a specific set of insects. The different entomopathogenic toxins of various Bt ssp. can be manipulated genetically in an educated way to construct more efficient transgenic bacteria or plants that express combinations of toxin genes to control pests.2 Joint research projects in our respective laboratories during the last decade demonstrate what can be done by implementing certain ideas using molecular biology with Bt ssp. israelensis (Bti) as a model system. Here, we describe our progress achieved with Gram-negative bacterial species, including cyanobacteria, and some preliminary experiments to form transgenic plants, mainly to control mosquitoes (Diptera), but also a particular Lepidopteran and Coleopteran pest species. In addition, a system is described by which environment-damaging genes can be removed from the recombinants thus alleviating procedures for obtaining permits to release them in nature.

In order to understand the influence of the 20 kDa protein on the cytolytic activity of CytA protein, a set of PCR primers were designed to amplify 20 kDa protein and CytA protein genes. The genes were ligated to E. coli expression vector pUHE24 and transformed into E. coli XL1 and DH5 alpha. The clones, LZ20, LZcytA and LZ20A containing 20 kDa protein gene, cytA protein gene and 20 kDa-cytA genes were obtained respectively. The influence of the clones on the growth of E. coli cells was determined under induction of IPTG. The results showed that the growth of LZ20 cells were not affected, LZcytA cells were killed, and the growth of LZ20A cells were not affected. It was suggested that expression product of 20 kDa protein gene protected the host cells from the cytolytic effect of CytA protein. This was supported by using different host strains.

Transcript levels of several Escherichia coli genes involved in chromosome replication and cell division were measured in dnaC2(Ts) mutants synchronized for chromosome replication by temperature shifts. Levels of transcripts from four of the genes, dam, nrdA, mukB, and seqA, were reduced at a certain stage during chromosome replication. The magnitudes of the decreases were similar to those reported previously ftsQ and ftsZ (P. Zhou and C. E. Helmstetter, J. Bacteriol. 176:6100-6106, 1994) but considerably less than those seen with dnaA, gidA, and mioC (P. W. Theisen, J. E. Grimwade, A. C. Leonard, J. A. Bogan, and C. E. Helmstetter, Mol. Microbiol. 10:575-584, 1993). The decreases in transcripts appeared to correlate with the estimated time at which the genes replicated. This same conclusion was reached in studies with synchronous cultures obtained with the baby machine in those instances in which periodicities in transcript levels were clearly evident. The transcriptional levels fo...

A large plasmid containing all delta-endotoxin genes was isolated from Bacillus thuringiensis subsp. israelensis; restricted by BamHI, EcoRI, HindIII, KpnI, PstI, SacI, and SalI; and cloned as appropriate libraries in Escherichia coli. The libraries were screened for inserts containing recognition sites for BamHI, SacI, and SalI. Each was labeled with 32P and hybridized to Southern blots of gels with fragments generated by cleaving the plasmid with several restriction endonucleases, to align at least two fragments of the relevant enzymes. All nine BamHI fragments and all eight SacI fragments were mapped in two overlapping linkage groups (with total sizes of about 76 and 56 kb, respectively). The homology observed between some fragments is apparently a consequence of the presence of transposons and repeated insertion sequences. Four delta-endotoxin genes (cryIVB-D and cytA) and two genes for regulatory polypeptides (of 19 and 20 kDa) were localized on a 21-kb stretch of the plasmid; ...

Lipophilic cations (tetraphenylarsonium, tetraphenylphosphonium, and triphenylmethylphosphonium) caused a number of major changes in the physiology of Bacillus subtilis. Macromolecular synthesis was inhibited, adenosine 5'-triphosphate concentration increased, swimming speed was reduced, tumbling was suppressed, and the capacity to take up the cations was greatly enhanced; respiration was not significantly altered. The effects occurred at lipophilic cation concentrations in the range commonly employed for measurement of membrane potential. Neither the enhancement of cation uptake nor the motility inhibition was a consequence of alteration of membrane potential, since both effects were still seen in the presence of valinomycin, with the extent of 86Rb+ uptake indicating a constant potential. Because suppression of tumbling accompanied speed reduction, as has also been found when protonmotive force is reduced, it is likely that lipophilic cations are perturbing the process of conv...

The transition from rod-shaped to spheroidal cells was studied in a temperature-sensitive strain (SP45) of Escherichia coli K12, carrying a mutation (pbpA) in the gene coding for penicillin-binding protein 2 (PBP-2). This transition imposed by the restrictive temperature was associated with reduction of peptidoglycan/surface area and of cellular osmotic stability. Addition of nalidixic acid (20 micrograms/ml) at the temperature shift from 30 to 42 degrees C resulted in lysis of some cells and appearance of spheroidal bulges along the cylinders in other cells, consistent with the hypothesis of envelope weakening due to inactivation of PBP-2.

Fluorescence polarization, P, of 1,6-diphenyl-1,3,5-hexatriene was studied in Escherichia coli B/r. Modification of nutritional conditions was not compensated by homeoviscous adaptation, demonstrated to exist for temperature variations. Cell diameter, which is known also to vary with nutrition but not with temperature, was found to be positively correlated with 1/P, and may therefore be regulated by membrane lipid order and fluidity.

Repetitive DNA is a periodic copolymer with the intrinsic property of exponential propagation to longer repeats. Microgene polymerization reaction (MPR) is a model system in which a short nonrepetitive homo-duplex DNA evolves to multiple repetitive products during heat-cool cycles. The mechanism underlying this process involves staggered annealing of complementary DNA strands of variable lengths and polymerase-mediated filling-in of the generated overhangs. MPR is considered here as a process sharing common features with two polymerization types, chain-growth and step-growth, and significant distinctions from both types were highlighted. The involved reaction stages were formulated and a kinetic model was derived and tested experimentally. The model can quantitatively explain MPR propagation and be used as a good approximation for this phenomenon.

Conventional methods often fail to control the flatheaded borers Capnodis spp., major pests of stone fruit trees; the larvae are protected from insecticides and predation because they feed deep in the roots. A potential solution is transgenic trees producing in their roots toxic compounds such as Cry proteins of Bacillus thuringiensis (Bt). Toxicities against Capnodis larvae were demonstrated by exploiting a recently designed artificial larval diet and an available collection of field isolated Bt. An isolate of Bt tenebrionis (Btt) from commercial bioinsecticide (Novodor) displayed LC50 and LC95 values of 3.2 and 164 mg g(-1) , respectively, against neonates of Capnodis tenebrionis, whereas values of the most toxic field isolate K-7 were 1.9 and 25.6 mg g(-1) respectively. Weights of surviving larvae after 1 month on diets containing low concentrations of K-7 (0.1-1.0 mg g(-1) ) were lower than on Btt or untreated larvae. K-7 was also toxic against larvae of C. cariosa and C. miliaris and found to harbour genes encoding Cry9Ea-like and Cry23Aa/Cry37Aa binary toxins. Larvae of Capnodis spp. are susceptible to Bt Cry toxins. Expressing cry genes active against these pests thus seems a feasible solution towards production of transgenic rootstock trees resilient to the pest.

... This article is dedicated to the memory of our colleague and friend, the late Professor Yoel Margalith. Page 2. ... This se-quence (named Δ cry11Bb2) was cloned for expression in Escherichiacoli at the NcoI-BamHI sites of pUHE-24S [Ben-Dov et al., 1995] to yield plasmid pHNt-B ...

Mosquito larvicidal activity of Bacillus thuringiensis subsp. israelensis (Bti) is contained in parasporal crystal composed of 5 major proteins (of 134, 128, 78, 72 and 27 kDa), encoded respectively by cry4Aa, cry4Ba, cry10Aa, cry11Aa and cyt1Aa, all reside on the 128 kbp plasmid pBtoxis [1]. Three (excluding cry4Ba and cry10Aa) have been cloned in Escherichia coli together with p20 (encoding an accessory protein) in all 15 possible combinations and express the genes included [2]. Two of these, expressing cyt1Aa, p20 and cry4Aa, with or without cry11Aa, display high toxicity against Aedes aegypti larvae. When all 4 genes were introduced into the nitrogen-fixing, filamentous cyanobacterium Anabaena PCC 7120 for expression under two strong promoters PpsbA and PA1 [3], it displayed highest toxicity ever achieved in transgenic cyanobacteria against 4th-instar larvae of A. aegypti. Cyt1Aa was found to synergize both Cry4Aa and Cry11Aa, and shorten the lethal times (killing quicker), whic...

Spores of Bacillus thuringiensis subsp. israelensis and their toxic crystals are bioencapsulated in the protozoan Tetrahymena pyriformis, in which the toxin remains stable. Each T. pyriformis cell concentrates the spores and crystals in its food vacuoles, thus delivering them to mosquito larvae, which rapidly die. Vacuoles containing undigested material are later excreted from the cells. The fate of spores and toxin inside the food vacuoles was determined at various times after excretion by phase-contrast and electron microscopy as well as by viable-cell counting. Excreted food vacuoles gradually aggregated, and vegetative growth of B. thuringiensis subsp. is- raelensis was observed afte r7ha sfilaments that stemmed from the aggregates. The outgrown cells sporulated between 27 and 42 h. The spore multiplication values in this system are low compared to those obtained in carcasses of B. thuringiensis subsp. israelensis-killed larvae and pupae, but this bioencapsulation represents a n...

Sixteen Escherichia coli clones were assayed against susceptible and Bacillus thuringiensis-resistant Culex quinquefasciatus larvae. The clones expressed different combinations of four genes from Bacillus thuringiensis ssp. israelensis; three genes encoded mosquitocidal toxins (Cry11Aa, Cry4Aa and Cyt1Aa) and the fourth encoded an accessory protein (P20). The cross-resistance spectra of the mosquitoes were similar to the profiles for recombinant B. thuringiensis strains expressing B. thuringiensis toxin genes, but with varied toxicity levels. The toxicity of the recombinants towards resistant mosquito larvae was improved when p20 and cyt1Aa were expressed in combination with cry4Aa and/or cry11Aa. Recombinant pVE4-ADRC, expressing cry4Aa, cry11Aa, p20 and cyt1Aa, was the most active against the resistant Culex, and resistance levels did not exceed fourfold. These results indicate that B. thuringiensis ssp. israelensis genes expressed in a heterologous host such as E. coli can be effective against susceptible and B. thuringiensis-resistant larvae and suppress resistance.

The bacterial biota in larvae of Capnodis tenebrionis, a serious pest of cultivated stone-fruit trees in the West Palearctic, was revealed for the first time using the MiSeq platform. The core bacterial community remained the same in neonates whether upon hatching or grown on peach plants or an artificial diet, suggesting that C. tenebrionis larvae acquire much of their bacterial biome from the parent adult. Reads affiliated with class levels Gammaproteobacteria and Alphaproteobacteria (phylum Proteobacteria ca. 86%), and Actinobacteria (ca. 14%) were highly abundant. Most diverse reads belong to the families Xanthomonadaceae (50%), Methylobacteriaceae (20%), Hyphomicrobiaceae (9%), Micrococcaceae (7%) and Geodermatophilaceae (4.5%). About two-thirds of the reads are affiliated with the genera Lysobacter, Microvirga, Methylobacterium, and Arthrobacter, which encompass species displaying cellulolytic and lipolytic activities. This study provides a foundation for future studies to elu...

Escherichia coli nucleoids were visualized after the DNA of OsO4-fixed but hydrated cells was stained with the fluorochrome DAPI (4',6-diamidino-2-phenylindole dihydrochloride hydrate). In slowly growing cells, the nucleoids are rod shaped and seem to move along the major cell axis, whereas in rapidly growing, wider cells they consist of two- to four-lobed structures that often appear to advance along axes lying perpendicular or oblique to the major axis of the cell. To test the idea that the increase in cell diameter following nutritional shift-up is caused by the increased amount of DNA in the nucleoid, the cells were subjected to DNA synthesis inhibition. In the absence of DNA replication, the nucleoids continued to move in the growing filaments and were pulled apart into small domains along the length of the cell. When these cells were then transferred to a richer medium, their diameters increased, especially in the region enclosing the nucleoid. It thus appears that the nuc...

It is crucial to the reproducibility of results and their proper interpretation that the conditions under which experiments are carried out be defined with rigour and consistency. In this review we attempt to clarify the differences and interrelationships among steady, balanced and exponential states of culture growth. Basic thermodynamic concepts are used to introduce the idea of steady-state growth in open, biological systems. The classical, sometimes conflicting, definitions of steady-state and balanced growth are presented, and a consistent terminology is proposed. The conditions under which a culture in balanced growth is also in exponential growth and in steady-state growth are indicated. It is pointed out that steady-state growth always implies both balanced and exponential growth, and examples in which the converse does not hold are described. More complex situations are then characterized and the terminology extended accordingly. This leads to the notion of normal growth and growth that can be synchronous or otherwise unbalanced but still reproducible, and to the condition of approximate steady state manifested by growth in batch culture and by asymmetrically dividing cells, which is analysed in some detail.