Electrical Characteristics of Triturus Egg Cells during Cleavage

The membrane potential in the blastomeres of dividing Triturus egg cells increases progressively from the first cleavage to the late morula stages. Both the animal and vegetal poles show the same increasing trend in potential; there is no significant potential difference between them. Upon first cell cleavage, the total resistance of the egg cell surface in contact with the exterior decreases to about one-tenth of its value before cleavage, and then remains rather constant up to the late morula stage. The specific resistance of this membrane surface drops rather abruptly upon first cleavage, and rises progressively during the morula stage. The resistance of the junctional membrane surface of the blastomeres, that is, the membrane formed at the former planes of cleavage, is small in relation to that of the cell surface in contact with the exterior. As a result, the blastomeres are electrically coupled throughout all stages of embryonic development examined.     

The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

Selection of amidases with novel substrate specificities from penicillin amidase of Escherichia coli

To obtain amidases with novel substrate specificity, the cloned gene for penicillin amidase of Escherichia coli ATCC 11105 was mutagenized and mutants were selected for the ability to hydrolyze glutaryl-(L)-leucine and provide leucine to Leu- host cells. Cells with the wild-type enzyme did not grow in minimal medium containing glutaryl-(L)-leucine as a sole source of leucine. The growth rates of Leu- cells that expressed these mutant amidases increased as the glutaryl-(L)-leucine concentration increased or as the medium pH decreased. Growth of the mutant strains was restricted by modulation of medium pH and glutaryl-(L)-leucine concentration, and successive generations of mutants that more efficiently hydrolyzed glutaryl-(L)-leucine were isolated. The kinetics of glutaryl-(L)-leucine hydrolysis by purified amidases from two mutants and the respective parental strains were determined. Glutaryl-(L)-leucine hydrolysis by the purified mutant amidases occurred most rapidly between pH 5 and 6, whereas hydrolysis by wild-type penicillin amidase at this pH was negligible. The second-order rate constants for glutaryl-(L)-leucine hydrolysis by two "second-generation" mutant amidases, 48 and 77 M-1 s-1, were higher than the rates of hydrolysis by the respective parental amidases. The increased rates of glutaryl-(L)-leucine hydrolysis resulted from both increases in the molecular rate constants and decreases in apparent Km values. The results show that it is possible to deliberately modify the substrate specificity of penicillin amidase and successively select mutants with amidases that are progressively more efficient at hydrolyzing glutaryl-(L)-leucine.     

Selection of amidases with novel substrate specificities from penicillin amidase of Escherichia coli.

To obtain amidases with novel substrate specificity, the cloned gene for penicillin amidase of Escherichia coli ATCC 11105 was mutagenized and mutants were selected for the ability to hydrolyze glutaryl-(L)-leucine and provide leucine to Leu- host cells. Cells with the wild-type enzyme did not grow in minimal medium containing glutaryl-(L)-leucine as a sole source of leucine. The growth rates of Leu- cells that expressed these mutant amidases increased as the glutaryl-(L)-leucine concentration increased or as the medium pH decreased. Growth of the mutant strains was restricted by modulation of medium pH and glutaryl-(L)-leucine concentration, and successive generations of mutants that more efficiently hydrolyzed glutaryl-(L)-leucine were isolated. The kinetics of glutaryl-(L)-leucine hydrolysis by purified amidases from two mutants and the respective parental strains were determined. Glutaryl-(L)-leucine hydrolysis by the purified mutant amidases occurred most rapidly between pH 5 and 6, whereas hydrolysis by wild-type penicillin amidase at this pH was negligible. The second-order rate constants for glutaryl-(L)-leucine hydrolysis by two "second-generation" mutant amidases, 48 and 77 M-1 s-1, were higher than the rates of hydrolysis by the respective parental amidases. The increased rates of glutaryl-(L)-leucine hydrolysis resulted from both increases in the molecular rate constants and decreases in apparent Km values. The results show that it is possible to deliberately modify the substrate specificity of penicillin amidase and successively select mutants with amidases that are progressively more efficient at hydrolyzing glutaryl-(L)-leucine.     

Differential expression of the three multicopper oxidases from Myxococcus xanthus

Myxococcus xanthus is a soil bacterium that undergoes a unique life cycle among the prokaryotes upon starvation, which includes the formation of macroscopic structures, the fruiting bodies, and the differentiation of vegetative rods into coccoid myxospores. This peculiarity offers the opportunity to study the copper response in this bacterium in two different stages. In fact, M. xanthus vegetative rods exhibit 15-fold-greater resistance against copper than developing cells. However, cells pre-adapted to this metal reach the same levels of resistance during both stages. Analysis of the M. xanthus genome reveals that many of the genes involved in copper resistance are redundant, three of which encode proteins of the multicopper oxidase family (MCO). Each MCO gene exhibits a different expression profile in response to external copper addition. Promoters of cuoA and cuoB respond to Cu(II) ions during growth and development; however, they show a 10-fold-increased copper sensitivity during development. The promoter of cuoC shows copper-independent induction upon starvation, but it is copper up-regulated during growth. Phenotypic analyses of deletion mutants reveal that CuoB is involved in the primary copper-adaptive response; CuoA and CuoC are necessary for the maintenance of copper tolerance; and CuoC is required for normal development. These roles seem to be carried out through cuprous oxidase activity.     

Variability of Flavonol Contents during Floral Morphogenesis in the Poppy Papaver somniferum L.

We studied the contents of flavonols (kaempferol and quercetin) in the meristem of vegetative and generative apices of the main plant shoot in floral Papaver somniferum L. mutants, as well as in the normal plants at successive stages of flower development. Five stages of flower development were distinguished. Flavonols (kaempferol and quercetin) were present in all flower organs at all stages of floral morphogenesis we studied. However, their contents and distribution in different organs and at different stages of flower development markedly varied. No significant differences were found in the contents of flavonols in the meristems of vegetative and generative apices of the main shoot in the lines of floral mutants, as well as between the lines with different amounts of vegetative phytomeres. In the plants with normal flower structure, the contents of flavonols (kaempferol + quercetin) sharply increased with the beginning of differentiation of flower organs, i.e. from stage 3, to reach a maximum in the open flower, when gametogenesis is terminated and fertilization takes place. The level of flavonol contents in the petals (upper part) and stamen was at a maximum at all stages of flower development, while that in the gynaecium was at a minimum. The kaempferol : quercetin ratio was shifted towards quercetin at successive stages of flower development, most significantly in the stamens. The involvement of flavonols in the regulation of floral morphogenesis at stages of flower organs differentiation and functioning is discussed.     

Genetic Studies of Acridine-Induced Mutants in STREPTOCOCCUS PNEUMONIAE

The mutagenic properties of acridines on pneumococcus are described. All seven acridines tested were mutagenic at the amiA locus conferring a resistance to 10^-5 m aminopterin. The effects of quinacrine were more specifically investigated. It was observed that: mutants can be obtained only by treatment of exponentially growing cells; a sharp maximum mutagenic effect occurs at a concentration slightly lower than the bacteriostatic value; and the amount of quinacrine required to yield the maximum mutagenic effect decreases with the pH of the medium. Moreover, the number of mutants detected after quinacrine treatment varies from locus to locus. The majority of quinacrine-induced mutants are readily reverted by quinacrine, but not by nitrosoguanidine treatment. This suggests that in pneumococcus quinacrine induces mainly frameshift mutations. A further study of the revertants obtained by quinacrine treatment of quinacrine-induced mutants strengthens this interpretation: most of the revertants result from a mutation at the same site; some partial revertants exhibiting an intermediate resistance to aminopterin were found to contain two very closely linked mutated sites, each mutation conferring the maximum level of resistance to aminopterin. Thus, the majority of quinacrine-induced mutants at the amiA locus of pneumococcus consists of frameshift mutations. Nearly all of the isolated mutants induced by quinacrine as well as other acridines belong to the low efficiency class of transformation. It was concluded that the mismatch resulting from the pairing between the wild type and the frameshift-containing sequence is recognized by the excision-repair system involved in the discrimination function in a way similar to that in which it recognizes mismatched base pairs between a transition mutation and the wild-type sequence.     

Variability of flavonol contents during floral morphogenesis in Papaver somniferum L

We studied the contents of flavonols (kaempferol and quercetin) in the meristem of vegetative and generative apices of the main plant shoot in floral Papaver somniferum mutants, as well as in the normal plants at successive stages of flower development. Five stages of flower development were distinguished. Flavonols (kaempferol and quercetin) were present in all flower organs at all stages of floral morphogenesis we studied. However, their contents and distribution in different organs and at different stages of flower development markedly varied. No significant differences were found in the contents of flavonols in the meristems of vegetative and generative apices of the main shoot in the lines of floral mutants, as well as between the lines with different amounts of vegetative phytomeres. In the plants with normal flower structure, the contents of flavonols (kaempferol + quercetin) sharply increased with the beginning of differentiation of flower organs, i.e. from stage 3, to reach a maximum in the open flower, when gametogenesis is terminated and fertilization takes place. The level of flavonol contents in the petals (upper part) and stamen was at a maximum at all stages of flower development, while that in the gynaecium was at a minimum. The kaempferol: quercetin ratio shifted towards quercetin at successive stages of flower development, most significantly in the stamens. The involvement of flavonols in the regulation of floral morphogenesis at stages of flower organs differentiation and functioning is discussed.     

Novel phenotypes and developmental arrest in early embryo specific mutants of maize

Embryo specific (emb) mutants exhibit aberrant embryo development without deleterious effects on endosperm development. We have analyzed five emb mutants of maize, which, based on their developmental profiles can be divided into two groups: mutants arrested at early stages and mutants with novel phenotypes. The members of the first group resemble wild-type proembryos and never reach other developmental stages. In the second group the tube-shaped mutants emb*-8522 and emb*-8535 completely lack apical-basal differentiation, while in mutant emb*-8516 a second embryo-like structure arises from the suspensor. The five emb mutations analyzed are non-allelic and two of the mutations are very likely caused by insertion of the transposon mutator, opening the door for their molecular analysis. Received: 10 February 1999 / Accepted: 7 July 1999     

Unstable ascospore color mutants of Ascobolus immersus

Summary In Ascobolus immersus, 16 unstable mutants with white ascospores were isolated. These mutants mutate back to the wild-type phenotype with brown ascospores. Only two mutants B and 301 are the object of the present study. At least in these two mutants it was demonstrated that true back-mutations occur. The frequencies of back-mutations are quite high and can reach 0.30 in the case of mutant B.The reversions appear at well defined but different for each mutant stages of the developmental cycle. These stages are relatively short and involve not more than about ten nuclear divisions cycles. In the case of mutant B, the back-mutations occur only in very young mycelia and at elevated temperature. Thus, the frequencies of back-mutations observed depend on the time of the transfer to higher temperature condition.The back-mutations of 301 mutant occur only in spermatized female organ and depend on the genotype of the female parent.The data established for Ascobolus immersus are compared with genic instability due to transposable elements described in corn by B. Mc Clintock.     

Short-term adaptation of microalgae in highly stressful environments: an experimental model analysing the resistance of Scenedesmus intermedius (Chlorophyceae) to the heavy metals mixture from the Aznalcóllar mine spill

The toxic spill of acid wastes rich in heavy metals/metalloids (AWHM) from the Aznalcóllar mine in April 1998, threatening the Doñana National Park, is considered to be the worst environmental disaster related to acute pollution in Spanish history. The aim of this work was to study the adaptation of microalgae (which play an important role as primary producers) from AWHM sensitivity to AWHM resistance by using the alga Scenedesmus intermedius as an experimental model. The Malthusian parameter (m) and the carrying capacity (K) were reduced by mud and soil samples collected from the affected area. A dose–effect analysis showed that fitness progressively diminished with increasing sample concentration. A fluctuation analysis demonstrated that AWHM-resistant cells arose by rare spontaneous mutations that occurred randomly prior to the incorporation of the AWHM. The rate of spontaneous mutation from AWHM sensitivity to AWHM resistance was 2·12×10^?5 mutants per cell division. A competition experiment between wild-type AWHM-sensitive cells and AWHM-resistant mutants showed that in small populations the AWHM-resistant mutants are driven to extinction in the absence of selection for AWHM resistance. The resistant phenotypes are maintained in the absence of AWHM as the result of a balance between spontaneous mutation and natural selection, so that about 43 AWHM-resistant mutants per million cells are present in the absence of AWHM. Our experimental model suggests that mutation is essential for adaptation of microalgal populations to environmental changes. Rare spontaneous pre-adaptive mutation is enough to ensure the survival of microalgal populations in contaminated environments when the population size is large enough.     

Photopigments in Rhodopseudomonas capsulata cells grown anaerobically in darkness.

The phototrophic bacterium Rhodopseudomonas capsulata can obtain energy for dark anaerobic growth from sugar fermentations dependent on accessory oxidants such as trimethylamine-N-oxide or dimethyl sulfoxide. Cells grown for one to two subcultures in this fashion, with fructose as the energy source, showed approximately a twofold increase in bacteriochlorophyll content (per milligram of cell protein) and developed extensive intracytoplasmic membranes in comparison with cells grown photosynthetically at saturating light intensity. Cells harvested from successive anaerobic dark subcultures, however, showed progressively lower pigment contents. After ca. 20 transfers, bacteriochlorophyll and carotenoids were barely detectable, and the amount of intracytoplasmic membrane diminished considerably. Spontaneous mutants incapable of producing normal levels of photosynthetic pigments arose during prolonged anaerobic dark growth. Certain mutants of this kind appear to have a selective advantage over wild-type cells under fermentative growth conditions. Of four pigment mutants characterized (two being completely unable to produce bacteriochlorophyll), only one retained the capacity to grow photosynthetically.     

Responses of plant vascular systems to auxin transport inhibition.

To assess the role of auxin flows in plant vascular patterning, the development of vascular systems under conditions of inhibited auxin transport was analyzed. In Arabidopsis, nearly identical responses evoked by three auxin transport inhibitor substances revealed an enormous plasticity of the vascular pattern and suggest an involvement of auxin flows in determining the sites of vascular differentiation and in promoting vascular tissue continuity. Organs formed under conditions of reduced auxin transport contained increased numbers of vascular strands and cells within those strands were improperly aligned. In leaves, vascular tissues became progressively confined towards the leaf margin as the concentration of auxin transport inhibitor was increased, suggesting that the leaf vascular system depends on inductive signals from the margin of the leaf. Staged application of auxin transport inhibitor demonstrated that primary, secondary and tertiary veins became unresponsive to further modulations of auxin transport at successive stages of early leaf development. Correlation of these stages to anatomical features in early leaf primordia indicated that the pattern of primary and secondary strands becomes fixed at the onset of lamina expansion. Similar alterations in the leaf vascular responses of alyssum, snapdragon and tobacco plants suggest common functions of auxin flows in vascular patterning in dicots, while two types of vascular pattern alterations in Arabidopsis auxin transport mutants suggest that at least two distinct primary defects can result in impaired auxin flow. We discuss these observations with regard to the relative contributions of auxin transport, auxin sensitivity and the cellular organisation of the developing organ on the vascular pattern.     

Sterol mutants of Neurospora crassa: their isolation, growth characteristics and resistance to polyene antibiotics

Summary A method is described for isolating sterol mutants of the filamentous fungus, Neurospora crassa. Most of the mutants carry gene mutations affecting the later stages of ergosterol biosynthesis and they accumulate other, as yet unidentified, sterol components but two mutants are blocked earlier in the pathway and respond to exogenous mevalonic acid. Altered sterol metabolism is associated with a reduced rate of growth, abnormal morphology, poor fertility and resistance to a variety of polyene antibiotics.     

PLEIOTROPIC REGULATORY LOCUS 2 exhibits unequal genetic redundancy with its homolog PRL1

In plants, signaling leading to resistance against biotrophic pathogens is complex. Perception of pathogenic microbes by resistance (R) proteins is relayed though successive activities of downstream components, in a network that is not well understood. PLEIOTROPIC REGULATORY LOCUS 1 (PRL1) and >20 other proteins are members of the MOS4-associated complex (MAC), a regulatory node in defense signaling. Of all characterized MAC members, mutations in PRL1 cause the most severe susceptibility towards both virulent and avirulent microbial pathogens. Genetic suppressors of prl1 represent new signaling elements and may aid in further unraveling of defense mechanisms. Our identification and characterization of a dominant suppressor of prl1 revealed a regulatory, gain-of-function mutation in PLEIOTROPIC REGULATORY LOCUS 2 (PRL2), a close homolog of PRL1. Loss-of-function mutants of PRL2 do not exhibit altered phenotypes; however, prl1 prl2 double mutants exhibit enhanced morphological defects consistent with unequal genetic redundancy between the homologs. Up-regulated gene expression mediated by the dominant prl2-1D allele completely suppresses disease susceptibility in the prl1 mutant background and also restores wild-type appearance, further supporting functional equivalence between the two PRL proteins.     

Antibiotic Resistant Group A Streptococci

A series of Streptococcus pyogenes strains, including strains isolated from patients, mutants which had acquired in vitro resistance to penicillin (Pc), mitomycin C (MC), tetracycline (TC) and chloramphenicol (CM), ultraviolet light induced α hemolytic mutants, as well as β hemolytic mutants (β mutants) derived from α hemolytic mutants (α mutants) were compared as to their antibiotic sensitivity, and physiological, biochemical and serological properties. To obtain β mutants from α mutants the following procedures were employed: (1) serial mouse passage, (2) serial serum-broth transfers, (3) cultivation in heat-killed cultures of parent strains, and (4) cultivation in broth containing bacterial DNA extracted from parent streptococcus cells. From the results obtained these strains could be divided into two major groups, each with two subgroups. Group 1 strains produce soluble hemolysins and are sensitive to Pc. Subgroup 1–1 strains are sensitive to other antibiotics too; subgroup 1–2 are resistant to certain antibiotics other than Pc, bacitracin and MC. Group 2 strains do not produce soluble hemolysins and resistant to Pc. Subgroup 2-1 strains are α hemolytic on horse blood agar and subgroup 2–2 are β hemolytic on the same medium. Pc resistance in group 2 strains was more than 100-fold higher than that of sensitive strains, and was accompanied by MC resistance, but to a lesser degree. Pc resistance in group 2 mutants could be induced by antibiotics other than Pc and also by ultraviolet irradiation. Although group 1 cells retained the characteristics of typical S. pyogenes, group 2 cells, both α and β hemolytic, lost most of the physiological, biochemical and serological properties of this species. The similarity of group 2 strains to group D or group N streptococcal strains in their general properties is discussed.     

Ultrastructure of stomatal development in Arabidopsis (Brassicaceae) leaves

Stomatal development was studied in wild-type Arabidopsis leaves using light and electron microscopy. Development involves three successive types of stomatal precursor cells: meristemoid mother cells, meristemoids, and guard mother cells (GMCs). The first two types divide asymmetrically, whereas GMCs divide symmetrically. Analysis of cell wall patterns indicates that meristemoids can divide asymmetrically a variable number of times. Before meristemoid division, the nucleus and a preprophase band of microtubules become located on one side of the cell, and the vacuole on the other. Meristemoids are often triangular in shape and have evenly thickened walls. GMCs can be detected by their roughly oval shape, increased starch accumulation, and wall thickenings on opposite ends of the cells. Because these features are also found in developing stomata, stomatal differentiation begins in GMCs. The wall thickenings mark the division site in the GMC since they overlie a preprophase band of microtubules and occur where the cell plate fuses with the parent cell wall. Stomatal differentiation in Arabidopsis resembles that of other genera with kidney-shaped guard cells. This identification of stages in stomatal development in wild-type Arabidopsis provides a foundation for the analysis of relevant genes and of mutants defective in stomatal patterning, cell specification, and differentiation.     

Colchicine resistant friend cells: Application to the study of actinomycin D induced erythroid differentiation

Colchicine resistant (Cch^R) mutants have been isolated from Friend erythroeleukemic cells by successive single-step selections. Measurements of the rate of uptake of [³H]-colchicine into whole cells, and the binding of [³H]-colchicine to cytoplasmic extracts, suggest that these mutants are colchicine-resistant due to a reduced membrane permeability to colchicine, rather than an altered intracellular colchicine-binding target. Consistent with this conclusion is the observation that non-toxic concentrations of Tween–80, a non-ionic detergent, potentiated colchicine uptake into mutant cells. In addition, these Friend cell mutants, like Cch^R mutants of other cell types, are cross-resistant to a variety of unrelated drugs, including daunomycin, puromycin, emetine, and actinomycin D. A comparison of the dose-response curves for the induction of Friend cell differentiation by actinomycin D of both wild-type and two Cch^R cells suggests that actinomycin D permeation is required for its effects on Friend cell differentiation. Potentiation of actinomycin D uptake by Tween–80 significantly lowered the concentration of drug required to induce hemoglobin synthesis in the Cch^R cells, but had no significant effect on either actinomycin D induction of Cch^S cells or DMSO induction of both Cch^S and Cch^R cells. In common with other chemical inducers of Friend cell differentiation, the addition of actinomycin D results in an early decrease in ⁸⁶ RbCl uptake, although this effect on transport occurred 14 hours later than that observed with DMSO.     

Replacement of the positively charged Walker A lysine residue with a hydrophobic leucine residue and conformational alterations caused by this mutation in MRP1 impair ATP binding and hydrolysis

MRP1 (multidrug resistance protein 1) couples ATP binding/hydrolysis at its two non-equivalent NBDs (nucleotide-binding domains) with solute transport. Some of the NBD1 mutants, such as W653C, decreased affinity for ATP at the mutated site, but increased the rate of ATP-dependent solute transport. In contrast, other NBD1 mutants, such as K684L, had decreased ATP binding and rate of solute transport. We now report that mutations of the Walker A lysine residue, K684L and K1333L, significantly alter the tertiary structure of the protein. Due to elimination of the positively charged group and conformational alterations, the K684L mutation greatly decreases the affinity for ATP at the mutated NBD1 and affects ATP binding at the unmutated NBD2. Although K684L-mutated NBD1 can bind ATP at higher concentrations, the bound nucleotide at that site is not efficiently hydrolysed. All these alterations result in decreased ATP-dependent solute transport to approx. 40% of the wild-type. In contrast, the K1333L mutation affects ATP binding and hydrolysis at the mutated NBD2 only, leading to decreased ATP-dependent solute transport to approx. 11% of the wild-type. Consistent with their relative transport activities, the amount of vincristine accumulated in cells is in the order of K1333L> or =CFTR (cystic fibrosis transmembrane conductance regulator)>K684L>wild-type MRP1. Although these mutants retain partial solute transport activities, the cells expressing them are not multidrug-resistant owing to inefficient export of the anticancer drugs by these mutants. This indicates that even partial inhibition of transport activity of MRP1 can reverse the multidrug resistance caused by this drug transporter.     

Ionophore-resistant mutants of Toxoplasma gondii reveal host cell permeabilization as an early event in egress

Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. Invasion and egress by this protozoan parasite are rapid events that are dependent upon parasite motility and appear to be directed by fluctuations in intracellular [Ca(2+)]. Treatment of infected host cells with the calcium ionophore A23187 causes the parasites to undergo rapid egress in a process termed ionophore-induced egress (IIE). In contrast, when extracellular parasites are exposed to this ionophore, they quickly lose infectivity (termed ionophore-induced death [IID]). From among several Iie(-) mutants described here, two were identified that differ in several attributes, most notably in their resistance to IID. The association between the Iie(-) and Iid(-) phenotypes is supported by the observation that two-thirds of mutants selected as Iid(-) are also Iie(-). Characterization of three distinct classes of IIE and IID mutants revealed that the Iie(-) phenotype is due to a defect in a parasite-dependent activity that normally causes infected host cells to be permeabilized just prior to egress. Iie(-) parasites underwent rapid egress when infected cells were artificially permeabilized by a mild saponin treatment, confirming that this step is deficient in the Iie(-) mutants. A model is proposed that includes host cell permeabilization as a critical part of the signaling pathway leading to parasite egress. The fact that Iie(-) mutants are also defective in early stages of the lytic cycle indicates some commonality between these normal processes and IIE.