Lambda red-mediated synthesis of plasmid linear multimers in Escherichia coli K12

Summary Expression of the red ⁺ and gam ⁺ genes of bacteriophage in plasmids cloned in Escherichia coli wild-type cells leads to plasmid linear multimer (PLM) formation. In mutants that lack exonuclease I (sbcB sbcC), either of these functions mediates PLM formation. In order to determine whether PLM formation in sbcB sbcC mutants occurs by conservative (break-join) recombination of circular plasmids or by de novo DNA synthesis, thyA sbcB sbcC mutants were transferred from thymine- to 5-bromo-2-deoxyuridine (BUDR)-supplemented medium, concurrently with induction of red ⁺ or gam ⁺ expression, and the density distribution of plasmid molecular species was analyzed. After a period of less than one generation in the BUDR-supplemented medium, most PLM were of heavy/heavy density. Circular plasmids, as well as chromosomal DNA, were of light/light or light/heavy density. These results indicate that Red or Gam activities mediate de novo synthesis of PLM in sbcB sbcC mutants. Examination of plasmid DNA preparations from sbcB sbcC mutants expressing gam ⁺ or red ⁺ reveals the presence of two molecular species that may represent intermediates in the PLM biosynthesis pathway: single-branched circles (-structures) and PLM with single-stranded DNA tails. While Gam-mediated PLM synthesis in sbcB mutants depends on the activity of the RecF pathway genes, Red-mediated PLM synthesis, like Red-mediated recombination, is independent of recA and recF activities. One of the red ⁺ products, protein, suppresses RecA deficiency in plasmid recombination and PLM synthesis in RecBCD^– Exol^– cells. The dependence of PLM synthesis on the RecE, RecF or Red recombination pathways and the dependence of plasmid recombination by these pathways on activities that are required for plasmid replication support the proposal that PLM synthesis and recombination by these pathways are mutually dependent. We propose the hypothesis that DNA double-stranded ends, which are produced in the process of PLM synthesis, are involved in plasmid recombination by the RecE, RecF and Red pathways. Conversely, recombination-dependent priming of DNA synthesis at 3 singles-tranded DNA ends is hypothesized to initiate PLM synthesis on circular plasmid DNA templates.Abbreviations PLM plasmid linear multimers - BUDR 5-bromo-2-deoxyuridine - bp base pair     

The comparative effects of a mono- and a bi-functional alkylating agent on recombination inChlamydomonas reinhardi

Summary Synchronous populations of diploid cells ofChlamydomonas reinhardi were exposed to a bifunctional (Nitrogen Mustard) and a monofunctional (Diethyl Sulphate) alkylating agent at various stages of the pre-meiotic interphase and meiosis, and the effect on recombination in the arg 1/pab 2 region of linkage group I assessed. Both compounds affected recombination—the monofunctional one depressing it during two short periods, one just pre-meiosis when the main DNA duplication for meiosis is thought to occur, and the other in early prophase. The bifunctional compound on the other hand depressed recombination over an extended period. These effects have been related to the ability of the agents used to affect DNA synthesis and the different temporal patterns of response interpreted in terms of differential repair activities. The results provide additional evidence of the existence of two periods during which recombination can be affected.     

Effect of hydroxyurea treatment on transmission and recombination of mitochondrial genes in Saccharomyces cerevisiae: a new method to modify the input of mitochondrial genes in crosses.

Summary The effects of hydroxyurea, an inhibitor of DNA synthesis, on the transmission and recombination of mitochondrial genes conferring resistance to different antibiotics in Saccharomyces cerevisiae have been studied.Under the conditions used hydroxyurea does not induce respiratory deficient mutants in treated cultures. Homosexual and heterosexual crosses have been performed in which one of the parents was grown for several generations in a medium containing 10^-1 M hydroxyurea prior to mating and the other parent was untreated. In all cases, the transmission of mitochondrial alleles originating from the hydroxyurea treated parent increased. In homosexual crosses the increase of transmission is coordinated for all the alleles. In heterosexual crosses, there is a differential increase of transmission of the different alleles depending on the distance of the marker considered from .All the observed effects can be easily interpreted according to the predictions of the model proposed by Dujon et al. (1974), if one assumes that hydroxyurea treatment increases the input of mitochondrial alleles of the treated parent without major modifications of the frequency of recombination. For each cross the relative copy number of mitDNA of the treated parent has been calculated and the increase so found is in good agreement with the modification of the overall frequency of recombinants observed. Effects of hydroxyurea on input can be interpreted if one assumes that hydroxyurea has a differential effect on mitochondrial and nuclear DNA synthesis: nuclear DNA synthesis would be inhibited while mitochondrial DNA synthesis would still continue.In conclusion, we propose a new powerful genetic tool for the study of the transmission and recombination of mitochondrial genes which offers the possibility of experimentally controlling input, without inducing rho^- mutation.     

Genetic studies on heterochromatin in Drosophila melanogaster and their implications for the functions of satellite DNA

In Drosophila melanogaster the centromeric heterochromatin of all chromosomes consists almost entirely of several different satellite DNA sequences. In view of this we have examined by genetic means the meiotic consequences of X chromosomes with partial deletions of their heterochromatin, and have found that the amount and position of recombination on each heterochromatically deleted X is substantially different from that of a normal X. It appears that the amount of heterochromatin is important in modifying the centromere effect on recombination. — In all the deleted Xs tested, chromosome segregation is not appreciably altered from that of a nondeleted control chromosome. Thus satellite DNA does not appear to be an important factor in determining the regular segregation of sex chromosomes in Drosophila. Additionally, since X chromosomes with massive satellite DNA deficiencies are able to participate in a chromocenter within salivary gland nuclei, a major role of satellite DNA in chromocenter formation in this tissue is also quite unlikely. — In order to examine the mechanisms by which the amount of satellite DNA is increased or decreased in vivo, we have measured cytologically the frequency of spontaneous sister chromatid exchanges in a ring Y chromosome which is entirely heterochromatic and consists almost exclusively of satellite DNA. In larval neuroblast cells the frequency of spontaneous SCE in this Y is approximately 0.3% per cell division. Since there is no meiotic recombination in D. melanogaster males and since meiotic recombination in the female does not occur in heterochromatin, our results provide a minimum estimate of the in vivo frequency of SCE in C-banded heterochromatin (which is predominantly simple sequence DNA), without the usual complications of substituted base analogs, incorporated radioactive label or substantial genetic content. — We emphasise that: (a) satellite DNA is not implicated in any major way in recognition processes such as meiotic homologue recognition or chromocenter formation in salivaries, (b) there is likely to be continuous variation in the amount of satellite DNA between individuals of a species; and (c) the amount of satellite DNA can have a crucial functional role in the meiotic recombination system.     

Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology

Summary To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between is pE194 derivatives carrying segments of the chromosomal -gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for -gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 by of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.     

Genetic exchanges caused by ultraviolet photoproducts in phage λ DNA molecules: The role of DNA replication

Summary Genetic recombination induced by structural damage in DNA molecules was investigated in E. coli K12 () lysogens infected with genetically marked phage . Photoproducts were induced in the phage DNA before infection by exposing them either to 313 nm light in the presence of acetophenone or to 254 nm light. To test the role of the replication of the damaged phage DNA on the frequency of the induced recombination, both heteroimmune and homoimmune crosses were performed.First, samples of a heteroimmune phage imm434 P80 exposed to these treatments were allowed to infect cells lysogenic for prophage cI857 P3. Phage DNA replication and maturation took place, and the resulting progeny phages were assayed for the frequency of P ⁺ recombinants. Recombination was less frequent in infected cells exposed to visible light and in wild type cells able to perform excision repair than in excision-defective lysogens. Therefore, much of the induced recombination can be atributed to the pyrimidine dimers in the phage DNA, the only photoproducts known to be dissociated by photoreactivating enzyme.Second, in homoimmune crosses, samples of similarly treated homoimmune P3 phages were allowed to infect lysogens carrying cI857 P80. Replication of the phage DNA containing ultraviolet photoproducts was repressed by immunity, and was futher blocked by the lack of the P gene product needed for replication. The lysogens were purified and scored for both colony forming ability and for P ⁺ recombinant prophages. The 254 nm photoproducts increased the frequency of recombination in these homimmune crosses, even though phage DNA replication was blocked. Irradiation with 313 nm light and acetophenone M, which produces dimers and unknown photoproducts, was not as effective per dimer as the 254 nm light.It is concluded from these results that certain unidentified 254 nm photoproducts can cause recombination even in the absence of DNA replication. They are not pyrimidine dimers, as they are not susceptible to excision repair or photoreactivation. In contrast, pyrimidine dimers appear to cause recombination only when the DNA containing them undergoes replication.     

Retardation of cell cycle progression in yeast cells recovering from DNA damage: A study at the single cell level

Summary Pedigree analyses of individual yeast cells recovering from DNA damage were performed and time intervals between morphological landmark events during the cell cycle (bud emergence and cell separation), were recorded for three generations. The associated nuclear behavior was monitored with the aid of DAPI staining. The following observations were made: (1) All agents tested (X-rays, MMS, EMS, MNNG, nitrous acid) delayed the first bud emergence after treatment, which indicates inhibition of the initiation of DNA replication. (2) Cells that survived X-irradiation progressed further through the cell cycle in a similar way to control cells. (3) Progress of chemically treated cells became extremely asynchronous because surviving cells stayed undivided for periods of varying length. (4) Prolongation of the time between bud emergence and cell separation was most pronounced for cells treated with the alkylating agents MMS and EMS. This is interpreted as retardation of ongoing DNA synthesis by persisting DNA adducts. (5) Cell cycle prolongation in the second and third generation after treatment was observed only with MMS treated cells. (6) In all experiments, individual cells of uniformly treated populations exhibited highly variable responses.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - EMS ethyl methanesulfonate - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Induction of transfer-cell formation by iron deficiency in the root epidermis of Helianthus annuus L.

Helianthus annuus L. responds to iron deficiency by forming a thickened cortex and abundant root hairs in a zone near the root apex that corresponds to the primary developmental stage. Cytological investigations revealed that within 24 to 48 h of iron deficiency most of the peripheral cells differentiate into transfer cells. The wall labyrinth is always situated on the peripheral walls that face the external medium. The cytoplasm of these cells is characterized by numerous mitochondria, extensive rough endoplasmic reticulum, and large leucoplasts containing protein bodies. These observations are discussed in relation to the fact that Helianthus, as an iron efficient plant, responds physiologically to iron deficiency by extrusion of H⁺, production of reducing substances, and a steep increase in the uptake efficiency of Fe.     

Metal binding drugs induce synthesis of four proteins in normal cells

The synthesis of four proteins in chick embryo cells in culture is induced by exposure to several metal binding drugs and several cations. We reported previously that two chelating agents, kethoxal bis(thiosemicarbazone) and disulfiram induce these proteins. We now extend these findings to include 8-hydroxyquinoline, ortho-phenanthroline, and thiosemicarbazide. The non-chelating analogues of these latter three compounds; 4-hydroxyquinoline, metaphenanthroline, and semicarbazide, do not induce. Other chelating agents, such as thenoyl trifluoroacetone, mercaptopyridine N-oxide, mercaptobenzothiazole, and methimazole induce. However, not all chelators induce since EDTA, penicillamine, α,α-dipyridyl, and several others are ineffective. Several cations, such as copper, zinc, cadmium, and mercuric ions induce, but cobalt, nickel, manganese, iron, lead, and platinum do not. The anions arsenite and arsenate induce, but bromide, dichromate, fluoride, metabisulfite, molybdate, nitrite, permanganate, phosphate, sulfite, and sulfate ions do not. The chelating agents that induce the proteins are ionophores for⁶⁴Cu. The chelating agents that do not induce, such as EDTA, penicillamine, and the nonchelating analogs of the inducers, are not ionophores for copper. Since arsenite induces but is not a copper ionophore, we hypothesize that the common mechanism for both these compounds and the inducing cations is an interaction with a sulfhydryl group site.     

Illegitimate recombination mediated by T4 DNA topoisomerase in vitro

Summary Illegitimate recombination dependent on T4 DNA topoisomerase in a cell-free system has recently been described. In that work, recombinants between two phage DNA molecules were produced by the topoisomerase alone, without an Escherichia coli extract. In this paper, it is shown that recombination between phage and circular plasmid DNA molecules can also be detected in the presence or absence of an E. coli extract but at frequencies two or three orders of magnitude lower than that observed in the phage-phage cross. The frequency is probably lower because multiple recombination is required in the case of the phage-plasmid cross.     

SSV1-encoded site-specific recombination system in Sulfolobus shibatae

Summary We present evidence for the existence of a conservative site-specific recombination system in Archaea by demonstrating integrative recombination of Sulfolobus shibatae virus SSV1 DNA with the host chromosome, catalysed by the SSVI-encoded integrase in vitro. The putative int gene of SSV1 was expressed in Escherichia coli yielding a protein of about 39 kDa. This protein alone efficiently recombined linear DNA substrates containing chromosomal (attA) and viral (attP) attachment sites; recombination with either negatively or positively supercoiled SSV1 DNA was less efficient. Intermolecular attA × attA and attP × attP recombination was also promoted by the SSV integrase. The invariant 44 by common attachment core present in all att sites contained sufficient information to allow recombination, whilst the flanking sequences effected the efficiency. These features clearly distinguish the SSV1 — encoded site — specific recombination system from others and make it suitable for the study of regulatory mechanisms of SSV1 genome — host chromosome interaction and investigations of the evolution of the recombination machinery.     

Ferredoxin degradation in growing Clostridium pasteurianum during periods of iron deprivation

Clostridium pasteurianum was grown in batch cultures on media with an initial iron concentration of 10 M. The uptake of iron and the synthesis of ferredoxin was followed. All the iron present in the medium was taken up by the cells before 50% of the final cell density was attained. The bacteria then continued to grow in the complete absence of exogenous iron. Ferredoxin was synthesized during growth until the exogenous iron concentration dropped below 1 M. During growth in the absence of iron ferredoxin was degraded with the result that at the end of growth the cells did not contain ferredoxin. The specific activity of the iron sulfur protein, pyruvate synthase (E.C. 1.2.7.1), remained constant during growth of C. pasteurianum in the absence of exogenous iron. This finding suggests that ferredoxin was used as an endogenous source of iron for the synthesis of essential iron proteins during periods of iron deprivation.The term ferredoxin degradation is used here to indicate that the ferredoxin content in the growing cells decreased more than could be accounted for by repeated cell division. Ferredoxin = holoferredoxin = protein containing iron and sulfide; apoferredoxin = protein free of iron and sulfide     

Impairment of <Emphasis Type="Italic">ntc</Emphasis>A gene revealed its role in regulating iron homeostasis,ROS production and cellular phenotype under iron deficiency in cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120

Iron deficiency ends up into several unavoidable consequences including damaging oxidative stress in cyanobacteria. NtcA is a global nitrogen regulator controls wide range of metabolisms in addition to regulation of nitrogen metabolism. In present communication, NtcA based regulation of iron homeostasis, ROS production and cellular phenotype under iron deficiency in Anabaena 7120 has been investigated. NtcA regulates the concentration dependent iron uptake by controlling the expression of furA gene. NtcA also regulated pigment synthesis and phenotypic alterations in Anabaena 7120. A significant increase in ROS production and corresponding reduction in the activities of antioxidative enzymes (SOD, CAT, APX and GR) in CSE2 mutant strain in contrast to wild type Anabaena 7120 also suggested the possible involvement of NtcA in protection against oxidative stress in iron deficiency. NtcA has no impact on the expression of furB and furC in spite of presence of consensus NtcA binding site (NBS) and ?10 boxes in their promoter. NtcA also regulates the thylakoid arrangement as well as related photosynthetic and respiration rates under iron deficiency in Anabaena 7120. Overall results suggested that NtcA regulates iron acquisition and in turn protect Anabaena cells from the damaging effects of oxidative stress induced under iron deficiency.     

A molecular model for conjugational recombination in Escherichia coli K12

Summary Conjugational recombination in Escherichia coli was investigated by measuring lacZ ⁺ product, -galactosidase, in crosses between lacZ mutants. Enzyme production in both Hfr and F-prime crosses was detected very soon after transfer of the donor lacZ allele. The level of enzyme activity was reduced by no more than two-fold when the recipient carried a recB mutation. With an F-prime donor, recombination appeared to be restricted largely to a short period immediately after transfer, with little evidence of recombination during subsequent exponential growth of the transconjugant cells. These observations are interpreted to suggest that recA dependent recombination is able to initiate with high efficiency at gaps present in the donor DNA before synthesis of a complementary strand is completed, and independently of recB function. A molecular model for conjugational recombination based on this idea is presented in terms of the known activities of recA and recBC products. Some of the predictions of the model are tested by analysing the recombinant genotypes produced in Hfr crosses with multiply marked strains.     

Indications about the mechanism of action and toxicity of netilmicin on the basis of the antiplastidial activity inEuglena gracilis

Summary The effects of the new antibiotic netilmicin (NT) were studied onEuglena gracilis green cells. It was found that, in the presence of the drug, chlorophyll synthesis was strongly inhibited and plastid structure dramatically altered as revealed by fluorescence and electron microscopic observations. Importantly, NT at low concentrations (10–20 g/ml) and for short periods of time (36–72 hours) induced a marked bleaching effect that was permanent and accompanied by the persistence in the colorless cells of poorly differentiated plastids. Other cell components were not influenced as shown by their appearance and also by the kinetics of growth and the cell viability. On the basis of the results and of the literature on the bleaching agents it is suggested that NT is an untoxic antibiotic which specifically inhibits protein synthesis at prokaryotic level.This work was supported by a grant from Italian Research Council (CNR), Contract N. 82.02016.04.     

Repair of DNA double-strand breaks in Escherichia coli K12 requires a functional recN product

Summary Mutation of the recN gene of Escherichia coli in a recBC sbcB genetic background blocks conjugational recombination and confers increased sensitivity to UV light and mitomycin C. The basis for this phenotype was investigated by monitoring the properties associated with recN mutations in otherwise wild-type strains. It was established that recN single mutants are almost fully resistant to UV irradiation, and that there is no detectable defect in repair of UV lesions by excision, error-prone, or recombinational mechanisms. However, recN mutations confer sensitivity to mitomycin C and ionizing radiation both in wild-type and recB sbcB strains. The sensitivity to ionizing radiation is correlated with a deficiency in the capacity to repair DNA double-strand breaks by a UV inducible mechanism. Recombinant phages that complement the recombination and repair defects of recN recBC sbcB mutants have been identified, and the recN gene has been cloned from these phages into a low copy-number plasmid.     

Specificity and mechanism of rhizoferrin-mediated metal ion uptake

Rhizoferrin-mediated iron uptake was studied in two different classes of organisms: a rhizoferrin producing fungus, Absidia spinosa (Zygomycetes), and a ferric rhizoferrin utilizing bacterium, Morganella morganii (Enterobacteriaceae). The uptake of iron rhizoferrin and some of its metal analogs (chromium, rhodium, gallium), was followed and kinetic parameters measured in A. spinosa. These metal ion complexes were taken up in a concentration- and energy-dependent manner indicative of an active transport system. The uptake of the kinetically inert chromium and rhodium and reductively inert gallium complexes suggests a variation of the so called shuttle mechanism may be operative. The recognition of one geometrical isomer of chromium-rhizoferrin but not another argues for a degree of stereospecificity in the uptake process. A growth promotion plate assay was used to examine metal-rhizoferrin uptake in M. morganii. The results indicate that a number of factors including the nature of the chelating agent (e.g. bipyridyl or EDDHA) used to induce iron deficiency need to be considered before these simple plate assays can be reliably used to indicate the presence or absence of a particular siderophore uptake system.     

Multiple sites of crossing over within the Eb recombinational hotspot in the mouse

The Eb gene of the mouse major histocompatibility complex (MHC) contains a well-documented hotspot of recombination. Twelve cases of intra-Eb recombination derived from the b, d, k and s alleles of the Eb gene were sequenced to more precisely position the sites of meiotic recombination. This analysis was based on positioning recombination breakpoints between nucleotide polymorphisms found in the sequences of parental haplotypes. All twelve cases of recombination mapped within the second intron of the Eb gene. Six of these recombinants, involving the k and s haplotypes, mapped to two adjoining DNA segments of 394 and 955 base pairs (bp) in the 3 half of the intron. In an additional two cases derived by crossing over between the d and s alleles, breakpoints were positioned to adjoining segments of 28 and 433 bp, also in the 3 half of the intron. Finally, four b versus k recombinants were mapped to non-contiguous segments of DNA covering 2.9 kb and 1005 bp of the intron. An analysis of the map positions of crossover breakpoints defined in this study suggests that the second intron of the Eb gene contains a recombinational hotspot of approximately 800–1000 bp which contains at least two closely linked recombinationally active sites or segments. Further examination of the sequence data also suggests that the postulated location for the recombinational hotspot corresponds almost precisely to an 812 bp sequence that shows nucleotide sequence similarity to the MT family of middle repetitive DNA.     

Role of homology and pathway specificity for recombination between plasmids and bacteriophage lambda

Summary To determine the minimum amount of homology required for efficient recombination in Escherichia coli, we measured recombination frequencies between bacteriophage and pBR322 derivatives containing DNA fragments of various sizes by assaying for phages that could transduce the bla and ori genes of pBR322. Efficient recombination required about 40 bp of homology; increases in homology above 40 bp resulted in proportionate increases in recombination, while decreases below 40 bp resulted in precipitous decreases in recombination. The recA ⁺ gene stimulated recombination over the entire range of homologies tested. Restriction enzyme digests of several recombinant DNA molecules indicated that they contained the complete plasmid DNA inserted in the genome as expected for a reciprocal crossover. Analysis of recombination frequencies in different recombination-deficient mutant strains indicated that the formation of -plasmid cointegrates by homologous recombination proceeded predominantly by the RecBC pathway and very inefficiently, if at all, by the RecE and RecF pathways.     

The effect of various chelating agents on the mobilization of iron from reticulocytes in the presence and absence of pyridoxal isonicotinoyl hydrazone

The chelating agent pyridoxal isonicotinoyl hydrazone (PIH) has recently been shown to mobilize ⁵⁹Fe from reticulocytes loaded with non-heme ⁵⁹Fe. In this study, various chelating agents were tested for their ability to effect the mobilization of iron from reticulocytes by PIH. They fall into several groups. The largest group includes chelators such as citrate, ethylenediaminetetracetic acid and desferrioxamine, which fail to affect PIH-induced iron mobilization and do not mobilize iron per se. Either these chelators do not enter reticulocytes or they do not take up iron from PIH-Fe complexes. The second group includes chelators such as 2,2′-bipyridine, 1,10-phenanthroline, bathophenanthroline sulfonate and N,N′-ethylenebis(o-hydroxyphenylglycine) which inhibit PIH-induced iron mobilization from reticulocytes and, when added together with PIH, induce radioiron accumulation in an alcohol-soluble fraction of reticulocytes. It appears that these chelators enter the cell and compete with PIH for ⁵⁹Fe(II), but having bound iron are unable to cross the cell membrane. Spectral analysis suggests that Fe(II) chelators such as 2,2′-bipyridine and 1,10-phenanthroline remove iron from Fe(II)PIH but are not able to do so from Fe(III)PIH. Then there are compounds such as 2,3-dihydroxybenzoic acid and catechol which potentiate PIH-induced iron mobilization although they are unable to mobilize iron from reticulocytes by themselves. Lastly, there is a group of miscellaneous compounds which include chelators that either potentiate the iron-mobilizing effect of PIH as well as mobilizing iron from reticulocytes by themselves (tropolone), or that reduce PIH-induced iron mobilization while themselves having an iron-mobilizing effect (N,N′-bis(2,3-dihydroxybenzoyl)-1,6-diaminohexane). In further experiments, heme was found to stimulate globin synthesis in reticulocytes, the heme synthesis of which was inhibited by PIH, suggesting that PIH is probably not toxic to the cells.