Cloning of the cysB gene of Erwinia carotovora subsp. carotovora, and the identification of its product

Summary The suicide vector pJB4JI was used to generate a range of Tn5-induced mutants of Erwinia carotovora subsp. carotovora (Ecc). One mutant, HC500, was a cysteine auxotroph which had a non-pectolytic, non-cellulolytic, non-proteolytic phenotype when grown under sulphate-limitation. The cysteine lesion of HC500 was shown to be analogous to the cysB mutation of Escherichia coli. The Ecc-cysB ⁺ gene product was identified as a protein of M_r 36000.     

Effect of ogt expression on mutation induction by methyl-, ethyl- and propylmethanesulphonate in Escherichia coli K12 strains

We have previously reported the isolation of an Escherichia coli K12 mutant that is extremely sensitive to mutagenesis by low doses of ethylating agents. We now show by Southern analysis that the mutation involves a gross deletion covering at least the ogt and fnr genes and that no O⁶-alkylguanine-DNA-alkyltransferase activity is present in cell-free extracts of an ada::Tn10 derivative of these bacteria. Confirmation that sensitisation to ethylation-induced mutagenesis was attributable to ogt and not to any other loci covered by the deletion was obtained by constructing derivatives. Thus an ogt::kan^r disruption mutation was introduced into the parental ogt ⁺ bacteria, and the ogt::kan^r mutation was then eliminated by cotransduction of ogt ⁺ with the closely linked Tet ^r marker (zcj::Tn10). The (ogt-fnr) deletion or ogt::kan^r disruption mutants were highly sensitive to ethyl methanesulphonate-induced mutagenesis, as measured by the induction of forward mutations to l-arabinose resistance (Ara¹). Furthermore, the number of Ara^r mutants increased linearly with dose, unlike the case in ogt ⁺ bacteria, which had a threshold dose below which no mutants accumulated. Differences in mutability were even greater with propyl methanesulphonate. Overproduction of the ogt alkyltransferase from a multicopy plasmid reduced ethylmethanesulphonate-induced mutagenesis in the ogt mutant strains and also methylmethanesulphonate mutagenesis in ada ^– bacteria. A sample of AB 1157 obtained from the E. coli K12 genetic stock centre also had a deletion covering the ogt and fnr genes. Since such deletions greatly influence the mutagenic responses to alkylating agents, a survey of the presence of the ogt gene in the E. coli K12 strain being used is advisable.     

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

Summary A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Km^r) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Km^r transfer frequency of 10^-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x10⁶ cpm for a cell density of 10³ colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.     

Induction of the Cysteine Regulon of Salmonella typhimurium in LB Medium Affects the Response of cysB Mutants to Mecillinam

Rapid growth of Salmonella typhimurium LT2 in rich LB-broth caused the induction of the cysteine regulon when the culture reached an optical density at 650 nm of 1.5. Addition of 0.05 mM L-cystine to LB-broth abolished induction, while 0.025 mM L-cystine delayed it for a doubling time (30 min). Induction did not occur when lack of oxygen or a temperature shift to 19°C slowed down growth in LB-broth. Uninduced cultures reached growth yield similar to that of induced cultures after overnight incubation. Growth on solid LB-medium also brought about induction, but to a lower level than in liquid medium. Leaky cysB mutants, which are sensitive to the β-lactam antibiotic mecillinam, displayed partial induction, whereas mecillinam-resistant cysB and cysE mutants showed no induction. It is suggested that induction develops in these mutants when constitutive transport systems fail to satisfy the high uptake of cysteine demanded by fast-growing cultures. The behavior of cysB mutants agrees with the hypothesis that, under some conditions, mecillinam action would be dependent on expression of the cysteine regulon. Received: 9 June 1999 / Accepted: 11 August 1999     

Analysis of the frequency of inheritance of transposed Ds elements in Arabidopsis after activation by a CaMV 35S promoter fusion to the Ac transposase gene

The Ac/Ds transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strep^r) progeny, but in many of these families a small proportion of strep^r seedlings were also resistant to hygromycin (hyg^r). Nevertheless, 70% of families tested did give rise to at least one strep^r, hyg^r seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep^rhyg^r progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strep^r seedlings were also hyg^r than after activation by 35S::TPase. We also examined the genotype of strep^r, hyg^r seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strep^r progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.     

The nifH, nifM and nifN genes of Azotobacter vinelandii: Characterisation by Tn5 mutagenesis and isolation from pLAFR1 gene banks

Summary Tn5 was introduced into Azotobacter vinelandii on a suicide vector, pGS9. Three Nif^- mutants were found to carry Tn5 in nifH (MV6), in nifN (MV22), and in or near nifM (MV21), from the results of hybridisation experiments. For MV21 and MV22 this was also shown by complementation with the nif genes of Klebsiella pneumoniae on pRD1. MV6 failed to synthesis the nifH, D and K gene products. MV6 and MV22 fixed nitrogen in the absence of supplied molybdenum while mutant MV21 did not, suggesting that the nifM gene product may be required for the alternative nitrogenase system synthesised in azotobacteria under conditions of molybdenum deprivation. Reconstitution experiments with mutant extracts showed that MV22 (nifN ^-) lacked the FeMo cofactor and that MV21 (NifM^-) synthesised inactive Fe protein. These biochemical phenotypes are identical to those of the K. pneumoniae nifN and nifM mutants, respectively, demonstrating that these genes have the same function in both K. pneumoniae and A. vinelandii. Complementation of the A. vinelandii mutants with pLAFR1 gene banks of A. vinelandii or a. chroococcum yielded three cosmids of interest. pLV10 complemented UW91, a nifH mutant, and corrected the defect in MV6 after recombination with the mutant genome. It also carried nifD (but not nifK) and about 18 kb of DNA upstream from nifH. pLV1 from the A. vinelandii gene bank complemented both MV21 and MV22 as did pLC11, isolated from the A. chroococcum gene bank. Both pLV1 and pLC11 carried part of the nif cluster downstream of nifHDK which also includes nifEN and nifMVS on about 22 kb of DNA.     

地毯草黄单胞菌双组分系统VgrS-VgrR基因敲除及表型筛选

【目的】研究地毯草黄单胞菌双组分系统VgrS-VgrR与致病性的关系,为木薯细菌性病害的高效防控提供分子生物学证据。【方法】采用同源重组方法构建vgrS和vgrR的插入失活突变体,用可移动的cosmid载体p HM1构建互补菌株。检测突变体的致病性、细菌游动性、胞外酶、胞外多糖的变化,观察细菌对H_2O_2和金属离子胁迫的反应。【结果】相比野生型菌株,vgrS和vgrR突变体接种寄主植物木薯后致病力显著降低,突变体的游动性减少、蛋白酶活性减弱、H_2O_2耐受性降低,在高浓度金属离子Fe²⁺、Fe³⁺、Cu²⁺、Ni²⁺、Zn²⁺、Co²⁺的胁迫条件下菌体生长显著减弱。然而,vgrS和vgrR突变体的胞外多糖含量显著升高,分别是野生型的2.14和1.89倍。【结论】阐明了VgrS-VgrR系统在细菌致病过程中发挥的重要作用,为鉴定VgrS-VgrR调控机制提供线索,为药物筛选提供靶向目标。     

The Salmonella typbimurium RecJ function permits growth of P22 abc phage on recBCD+ hosts

Summary We describe recJ mutants of Salmonella typhimurium. The recJ gene maps between sufD and serA (min 62) and is transcribed counterclockwise. Unlike recJ mutants of Escherichia coli, recJ strains of S. typhimurium are sensitive to irradiation with UV light. This sensitivity is equivalent to or greater that that displayed by recBCD mutant strains. The residual ability of phage P22 abc (anti-recBCD) mutants to form plaques on recBCD ⁺ strains is eliminated in recJ hosts. Thus host RecJ function appears to substitute for the anti-RecBCD functions of phage P22 and may serve to limit RecBCD activity.     

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.     

The nifU, nifS and nifV gene products are required for activity of all three nitrogenases of Azotobacter vinelandii

Summary Strains with mutations in 23 of the 30 genes and open reading frames in the major nif gene cluster of A. vinelandii were tested for ability to grow on N-free medium with molybdenum (Nif phenotype), with vanadium (Vnf phenotype), or with neither metal present (Anf phenotype). As reported previously, nifE, nifty, nifU, nifS and nifV mutants were Nif^– (failed to grow on molybdenum) while nifM mutants were Nif^–, Vnf^– and Anf^–. nifV, nifS, and nifU mutants were found to be unable to grow on medium with or without vanadium, i.e. were Vnf^– Anf–. Therefore neither vnf nor anf analogoues of nifU, nifS, nifV or nifM are expected to be present in A. vinelandii.     

Resistance to amino acid inhibition in Caulobacter crescentus

Summary Growth of Caulobacter crescentus CB15 in minimal glucose-ammonium media is inhibited by nine L-amino acids: cysteine, histidine, isoleucine, leucine, methionine, phenylalanine, serine, threonine, and valine. Addition of 10 mM L-alanine prevents the inhibitory response. Several other amino acids also prevented the inhibitory response but to a lesser extent. Mutants resistant to amino acid inhibition designated Aar^-, were isolated and characterized. All mutants exhibited a phenotype of cross-resistance to all amino acids and most analogues, regardless of the selective agent. Representative mutations were linked 80% to cysB by RP4-mediated conjugation, and all mutations were linked 10–25% to cysB by transduction. Although the mechanism of amino acid inhibition is not known, the alanine protection and the Aar^- phenotype seem to be due to a reduced rate of uptake of inhibitory amino acids.     

Characterization of the ntrBC genes of Azospirillam brasilense Sp7: Their involvement in the regulation of nitrogenase synthesis and activity

A 7.1 kb EcoRI fragment from Azospirillum brasilense, that hybridized with a probe carrying the ntrBC genes from Bradyrhizobium japonicum, was cloned. The nucleotide sequence of a 3.8 kb subfragment was established. This led to the identification of two open reading frames, encoding polypeptides of 401 and 481 amino acids, that were similar to NtrB and NtrC, respectively. A broad host range plasmid containing the putative Azospirillum ntrC gene was shown to restore nitrogen fixation under free-living conditions to a ntrC-Tn5 mutant of Azorhizobium caulinodans. Several Tn5 insertion mutants were isolated in the ntrBC coding region in A. brasilense. These mutants were prototrophic and Nif⁺. However, their nitrogenase activity was slightly lower than in the wild type and they were unable to grow on nitrate as sole nitrogen source. Under microaerobiosis and in the absence of ammonia, a nifA-lacZ fusion was expressed in the mutants at about 60% of the level in the wild type. In the presence of ammonia, the fusion was similarly expressed (60% of the maximum) both in the wild type and mutants. Addition of ammonia to a nitrogen-fixing culture of ntrBC mutants did not abolish nitrogenase activity, in contrast with the wild type. It thus appears that in Azospirillum the ntrBC genes are not essential for nitrogen fixation, although NtrC controls nifA expression to some extent. They are, however, required for the switch-off of nitrogenase activity.     

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: Insertion elements dominate the spontaneous spectra

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for α-complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-α, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

General organization of the genes specifically involved in the diaminopimelate-lysine biosynthetic pathway of Corynebacterium glutamicum

Summary To obtain animal cell lines carrying nonsense mutations and the corresponding suppressors, we used a supersuppressor selection strategy on the CHO cell line. The wild-type strain is resistant to the aminopterin present in HAT medium (i.e., it is HAT^r) because it contains the enzymes hypoxanthine-guanine phosphoribosyl transferase (HPRT) and thymidine kinase (TK), whereas both HPRT mutants — selected by their resistance to 6-thioguanine (TG^r) — and TK^- mutants — selected by their resistance to 5-bromodeoxyuridine (BrdUrd^r) — are HAT^s. Therefore, from HPRT^- TK^- double nonsense mutants, whose phenotype would be TG^r BrdUrd^r (HAT^s), simultaneous HPRT⁺ TK⁺ double phenotypic revertants could be obtained by selecting HAT^r (TG^s BrdUrd^s) variants carrying the corresponding nonsense supersuppressors. Through ethylmethane sulfonate (EMS) mutagenesis of the CHO cell line we obtained 65 TG^r variants, 53 of which were HAT^s and the rest HAT^r. Among 36 TG^r (HAT^s) variants tested, 23 did not revert to HAT^r, 4 reverted spontaneously and with EMS, and 9 reverted only with EMS. Some of the latter were probably HPRT^- nonsense mutants because they were very stringent (had less than 2% of wild-type [³H]hypoxanthine incorporation and HPRT enzyme activity), and did not complement genetically. The introduction of a second marker (BrdUrd^r) in 7 of these strains allowed us to isolate 29 TG^r BrdUrd^r (HAT^s) double drug-resistant lines. Through one-step mutagenesis and selection in HAT medium, from two double resistant strains we could isolate HAT^r (TG^s BrdUrd^s) wild-type phenotypic revertants, each of which probably carries suppressible HPRT and TK nonsense (or missense) alleles and the corresponding supersuppressor. Our strategy could now be extended to obtain variants carrying suppressors in other cell lines.     

Virulence genes are carried by a megaplasmid of the plant pathogen Pseudomonas solanacearum

Summary A class of avirulent mutants of the plant pathogenic bacterium Pseudomonas solanacearum, strain GMI1000, resistant to acridine orange (Acr^r), harbour a deletion of over 85 kb in their genome. This deletion affects, a1,000 kb megaplasmid which has previously been shown to be present in most of the strains of this species. In addition at least 11 out of 13 independent Tn5 insertions, leading to loss of virulence, are located on the megaplasmid. Nine of them are present in the region which is deleted from the Acr^r mutants. These results suggest that the majority of virulence genes identified so far are plasmid borne.     

Antibiotic production improvement in the rare actinomycete Planobispora rosea by selection of mutants resistant to the Aminoglycosides Streptomycin and Gentamycin and to Rifamycin

During a strain improvement program, spontaneous mutants with single or combined resistance to streptomycin (Str^r), gentamycin (Gen^r) or rifamycin (Rif^r) were selected from the industrial strain of Planobispora rosea, which is the producer of thiazolylpeptide GE2270. Among the mutants resistant to each single antibiotic, higher producers occurred more frequently (60%) among Gen^r than in Rif^r (10%) and Str^r (24%) populations. Two Gen^r mutants showed up to 1.5-fold improvement in GE2270 production while single resistant mutants Str^r and Rif^r produced slightly more than the parental strains. The combination of Str^r and Rif^r in the same strain improved GE2270 yield up to 1.7-fold. Finally, a higher GE2270 producing strain (1.8-fold improvement with respect to the parental strain) was selected among those mutants with triple resistance to streptomycin, rifamycin and gentamycin. A hierarchical increase in aerial mycelium and spore formation was observed which paralleled GE2270 production improvement.     

Using reverse micelles as microreactor for hydrogen production by coupled systems of Nostoc / R. palustris and Anabaena / R. palustris

Uptake hydrogenase negative mutants of bloom forming cyanobacteria (Nostoc and Anabaena) and the fermentative bacteria Rhodopseudomonas palustris P₄ were used together for producing hydrogen within the reverse micelles fabricated by N-ethyl hexyl sodium sulfosuccinate (AOT) in isooctane and cetyl trimethyl ammonium bromide (CTAB) in benzene. The rate of H₂ production in AOT/isooctane reverse micellar system was found to be more promising in comparison to the CTAB/Benzene reverse micellar entrapment. After mutagenesis in 2.0% (v/v) ethyl methane sulphonate (EMS) mutants of Nostoc and Anabaena were selected on BG-11 plates (containing 2% agar) and then used for analysis of produced hydrogen. In comparison to the unmutated Nostoc with R. palustris (within AOT/isooctane) the coupled system of mutated Nostoc and R. palustris produced H₂ by 3.9-fold higher rate, which is 8.6 mmol H₂/h/mg protein. Whereas, mutated Anabaena coupled with R. palustris produced 4.8 times higher hydrogen production within (AOT)/isooctane reverse micelles in comparison to the unmutated Anabaena with R. palustris. Effect of nitrogen to carbon ratio (N/C) on hydrogen production was studied and Anabaena/R. palustris and Nostoc/R. palustris systems were, respectively, found to generate 11.2 and 9.8 mmol H₂/h/mg protein continuously for 3 days. Effects of temperature and light intensity were also investigated and we found that 32°C temperature and 1,000 Lux light intensity are the optimum values in these systems. Addition of sodium dithionite also resulted in further enhancement of the rate and duration of hydrogen production in both (mutated Nostoc/R. palustris and mutated Anabaena/R.␣palustris) systems.     

Toxicity of and mutagenesis by chlorate are independent of nitrate reductase activity in Chlamydomonas reinhardtii

Summary Spontaneous chlorate-resistant (CR) mutants have been isolated from Chlamydomonas reinhardtii wildtype strains. Most of them, 244, were able to grow on nitrate minimal medium, but 23 were not. Genetic and in vivo complementation analyses of this latter group of mutants indicated that they were defective either at the regulatory locus nit-2, or at the nitrate reductase (NR) locus nit-1, or at very closely linked loci. Some of these nit-1 or nit-2 mutants were also defective in pathways not directly related to nitrate assimilation, such as those of amino acids and purines. Chlorate treatment of wild-type cells resulted in both a decrease in cell survival and an increase in mutant cells resistant to a number of different chemicals (chlorate, methylammonium, sulphanilamide, arsenate, and streptomycin). The toxic and mutagenic effects of chlorate in minimal medium were not found when cells were grown either in darkness or in the presence of ammonium, conditions under which nitrate uptake is drastically inhibited. Chlorate was also able to induce reversion of nit ^– mutants of C. reinhardtii, but failed to produce His ⁺ revertants or Ara^r mutants in the BA-13 strain of Salmonella typhimurium. In contrast, chlorate treatment induced mutagenesis in strain E1F1 of the phototrophic bacterium Rhodobacter capsulatus. Genetic analyses of nitrate reductase-deficient CR mutants of C. reinhardtii revealed two types of CR, to low (1.5 mM) and high (15 mM) chlorate concentrations. These two traits were recessive in heterozygous diploids and segregated in genetic crosses independently of each other and of the nit-1 and nit-2 loci. Three her loci and four lcr loci mediating resistance to high (HC) and low (LC) concentrations of chlorate were identified. Mutations at the nit-2 locus, and deletions of a putative locus for nitrate transport were always epistatic to mutations responsible for resistance to either LC or HC. In both nit ⁺ and nit ^– chlorate-sensitive (CS) strains, nitrate and nitrite gave protection from the toxic effect of chlorate. Our data indicate that in C. reinhardtii chlorate toxicity is primarily dependent on the nitrate transport system and independent of the existence of an active NR enzyme. At least seven loci unrelated to the nitrate assimilation pathway and mediating CR are thought to control indirectly the efficiency of the nitrate transporter for chlorate transport. In addition, chlorate appears to be a mutagen capable of inducing a wide range of mutations unrelated to the nitrate assimilation pathway.     

Genetic interactions and dosage effects of Polycomb group genes of Drosophila

The Polycomb (Pc) group of genes are required for maintenance of cell determination in Drosophila melanogaster. At least 11 Pc group genes have been described and there may be up to 40; all are required for normal regulation of homeotic genes, but as a group, their phenotypes are rather diverse. It has been suggested that the products of Pc group genes might be members of a heteromeric complex that acts to regulate the chromatin structure of target loci. We examined the phenotypes of adult flies heterozygous for every pairwise combination of Pc group genes in an attempt to subdivide the Pc group functionally. The results support the idea that Additional sex combs (Asx), Pc, Polycomblike (Pcl), Posterior sex combs (Psc), Sex combs on midleg (Scm), and Sex combs extra (Sce) have similar functions in some imaginal tissues. We show genetic interactions among extra sex combs (esc) and Asx, Enhancer of Pc, Pcl, Enhancer of zeste E(z), and super sex combs and reassess the idea that most Pc group genes function independently of esc. Most duplications of Pc group genes neither exhibit anterior transformations nor suppress the extra sex comb phenotype of Pc group mutations, suggesting that not all Pc group genes behave as predicted by the mass-action model. Surprisingly, duplications of E(z) enhance homeotic phenotypes of esc mutants. Flies with increasing doses of esc ⁺ exhibit anterior transformations, but these are not enhanced by mutations in trithorax group genes. The results are discussed with respect to current models of Pc group function.