The inhibitor peptide of the mitochondrial F1 · F0-ATPase interacts with calmodulin and stimulates the calmodulin-dependent Ca2+-ATPase of erythrocytes

The binding of calmodulin to the mitochondrial F₁ · F₀-ATPase has been studied. [^125I]Iodoazidocalmodulin binds to the ε-subunit and to the endogeneous ATPase inhibitor peptide in a Ca²⁺-dependent reaction. The effect of the mitochondrial ATPase inhibitor peptide on the purified Ca²⁺-ATPase of erythrocytes has also been analyzed. The inhibitor peptide stimulates the ATPase when pre-incubated with the enzyme. The activation of the Ca²⁺-ATPase by calmodulin is not influenced by the inhibitor peptide, indicating that the two mechanisms of activation are different. These in vitro effects of the two regulatory proteins may reflect a common origin of the two ATPases considered and/or of the regulatory proteins.     

Immunological detection of the mitochondrial F1-ATPase alpha subunit in the matrix of rat liver peroxisomes. A protein involved in organelle biogenesis?

Rat liver peroxisomes contain in their matrix the alpha-subunit of the mitochondrial F1-ATPase complex. The identification of this protein in liver peroxisomes has been achieved by immunoelectron microscopy and subcellular fractionation. No beta-subunit of the mitochondrial F1-ATPase complex was detected in the peroxisomal fractions obtained in sucrose gradients or in Nycodenz pelletted peroxisomes. The consensus peroxisomal targeting sequence (Ala-Lys-Leu) is found at the carboxy terminus of the mature alpha-subunit from bovine heart and rat liver mitochondria. Due to the dual subcellular localization of the alpha-subunit and to the structural homologies that exist between this protein and molecular chaperones [(1990) Biol. Chem. 265, 7713-7716] it is suggested that the protein should perform another functional role(s) in both organelles, plus to its characteristic involvement in the regulation of mitochondrial ATPase activity.     

Amino acid substitutions in the ε-subunit of the F1F0-ATPase of Escherichia coli

A mutant strain of Escherichia coli was isolated in which Gly-48 of the mature ε-subunit of the energy-transducing adenosine triphosphatase was replaced by Asp. This amino acid substitution caused inhibition of ATPase activity (about 70%), loss of ATP-dependent proton translocation and lowered oxidative phosphorylation, but did not affect proton translocation through the F₀. Purified F₁-ATPase from the mutant strain bound to stripped membranes with the same affinity as the normal F₁-ATPase. Partial revertant strains were isolated in which Pro-47 of the ε-subunit was replaced by Ser or Thr. Pro-47 and Gly-48 are predicted to be residues 2 and 3 in a Type II β-turn and the Gly-48 to Asp substitution is predicted to cause a change from a Type II to a Type I or III β-turn. Space-filling models of the β-turn (residues 46–49) in the normal, mutant and partial revertant ε-subunits indicate that the peptide oxygen between Pro-47 and Gly-48 is in a different position to the peptide oxygen between Pro-47 and Asp-48 and that the substitution of Pro-47 by either Ser or Thr restores an oxygen close to the original position. It is suggested that the peptide oxygen between Pro-47 and Gly-48 of the ε-subunit is involved either structurally in inter-subunit H-bonding or directly in proton movements through the F₁-ATPase.     

Does 2-hydroxy-5-nitrobenzyl bromide react with the epsilon-subunit of the mitochondrial F1-ATPase?

The incubation of bovine mitochondrial F1-ATPase with 2-hydroxy-5-nitrobenzyl bromide (HNB), a selective reagent toward tryptophan residues in proteins, produced a concentration dependent inactivation of the enzyme and the covalent binding of 0.88 mol reagent/mol F1. Although HNB is highly specific for tryptophan it has also some reactivity toward cysteine, then a pre-treatment of F1 with several sulphydryl reagents has been performed to make the site of reaction clearer. This pre-treatment had neither effects in the binding stoichiometry nor in the extent of catalytic inhibition, suggesting that readly accessible thiol groups are not involved in the reaction with HNB. Since the only tryptophan bearing polypeptide of the bovine mitochondrial F1-ATPase complex is its smallest subunit, subunit-epsilon, this is the most probable candidate for HNB reaction. Therefore it may be inferred that the intactness and/or the correct conformation of this subunit could be important factor(s) for the multisite ATP hydrolytic activity of the enzyme.     

Cyclic AMP-dependent phosphorylation of the precursor to beta subunit of mitochondrial F1-ATPase: a physiological mistake?

By using the purified rat liver protein for reference in electrophoresis and peptide mapping experiments, I have identified the beta subunit of mitochondrial F1-ATPase and its cytoplasmic precursor in two-dimensional gel patterns of proteins from S49 mouse lymphoma cells. The beta subunit precursor is a substrate for cAMP-dependent phosphorylation during its synthesis. Normally, both nonphosphorylated and phosphorylated forms of beta subunit precursor are processed rapidly to the smaller, more acidic forms of mature beta subunit. When processing is inhibited with valinomycin, both nonphosphorylated and phosphorylated forms of beta subunit precursor are stabilized. Nonphosphorylated beta subunit is one of the most stable of cellular proteins, but the phosphorylated form is eliminated within minutes of processing. This suggests that phosphorylated beta subunit is recognized as aberrant and excluded from assembly into the ATPase complex. These results argue that cAMP-dependent phosphorylation of the beta subunit precursor is a physiological mistake that is remedied after mitochondrial import and processing.     

Osmosensitivity of the 2H+−K+-exchange and the H+-ATPase complex F0F1 in anaerobically grownEscherichia coli

Escherichia coli grown anaerobically for osmotic studies upon increased osmolarity in alkaline medium carried out H⁺–K⁺-exchange in two steps, the first of which was DCCD¹ sensitive and osmo-dependent and had the 2H⁺/K⁺ stoichiometry. H⁺-efflux in the presence of protonophore (CCCP) upon increase of osmolarity was shown to be high and inhibited by DCCD, whereas H⁺-efflux induced by a decrease of osmolarity was small and not inhibited by DCCD. The 2H⁺/K⁺-exchange was absent intrkA anduncA mutants. InuncB mutant 2H⁺/K⁺-exchange was not DCCD-and osmosensitive. Competition between DCCD and osmoshock on inhibition of 2H⁺/K⁺-exchange was found. Osmosensitivity of this exchange disappeared in spheroplasts. Osmosensitivity of both 2H⁺/K⁺-exchange and the F₀F₁ and osmoregulation of the F₀F₁ via F₀ and a periplasmic space are postulated.Abbreviations F₀F₁ H⁺-ATPase complex - F₀ H⁺-channel, proteolipid - F₁ H⁺-ATPase - Trk constitutive system for K⁺ uptake - PV periplasmic protein valve - DCCD N,N-dicyclohexylcarbodiimide - CCCP carbonylcyanide-m-chlorophenylhydrazone - _H or _K transmembrane electrochemical gradient for H⁺ or K⁺ respectively - membrane potential - upshock or downshock increase or decrease of medium osmolarity, respectively - CGSC E. coli Genetic Stock Center, Yale University, USA     

Course of meiosis in F1 interspecific hybrids of Lycopersicon esculentum Mill. × Licopersicon chilense Dun.

A study was conducted into the course of meiosis in F₁ interspecific hybrids of Lycopersicum esculentum Mill (mutant line Mo 638) × Lycopersicum chinense Dul. and its parental forms. An F₁ interspecific hybrid was obtained through the embryo culture technique. A decrease in the chiasma frequency and an increase in the frequency of univalents and meiotic abnormalities compared to their parental forms were detected in hybrid plants. The number of univalents and the percentage of main impairments decreased, as the height of bud tier locations increased. A conclusion was made regarding the connection between the regularity of meiosis in the examined F₁ interspecific hybrids of Lycopersicon esculentum × Lycopersicon chilense, on the one hand, and the hybrid nature of genotypes and the influence of environmental factors, on the other hand.     

Does pyrophosphate bind to the catalytic sites of mitochondrial F1-ATPase?

The interactions between the pyrophosphate (PPi) binding sites and the nucleotide binding sites on mitochondrial F1-ATPase have been investigated, using F1 preparations containing different numbers of catalytic and noncatalytic nucleotide-binding sites occupied by ligands. In all cases, the total number of moles of bound nucleotides and PPi per mole of F1 was less than or equal to six. F1 preparations containing either three or two filled noncatalytic sites and no filled catalytic sites (referred as F1[3,0] and F1[2,0]) were found to bind 3 mol of PPi/mol of F1. Tight binding of ADP-fluoroberyllate complexes to two of the catalytic sites of F1 converted the three heterogeneous PPi-binding sites into three homogeneous binding sites, each exhibiting the same affinity for PPi. The addition of PPi at saturating concentrations to F1 containing GDP bound to two catalytic sites (F1[2,2]) resulted in the release of 1 mol of GDP. Furthermore, the addition of PPi to F1 filled with ADP-fluoroberyllate at the catalytic sites resulted in the release of 1 mol of tightly bound ADP/mol of F1. Taken together, these results indicate that PPi binds to specific sites that interact with both the catalytic and the noncatalytic nucleotide-binding sites of F1.     

Suppression of ρ 0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0

Specific mgi mutations in the α, β or γ subunits of the mitochondrial F₁-ATPase have previously been found to suppress ρ⁰ lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F₁ is dependent on the F₀ sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, ρ⁰ mutants can be isolated, upon treatment with ethidium bromide, that lack six major F₀ subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of ρ⁰ mutants indicates that an F₁-complex carrying a mgi mutation can assemble in the absence of F₀ subunits and that suppression of ρ⁰ lethality is an intrinsic property of the altered F₁ particle.     

Protein Kinase C-α Interaction with F0F1-ATPase Promotes F0F1-ATPase Activity and Reduces Energy Deficits in Injured Renal Cells

We showed previously that active PKC-α maintains F₀F₁-ATPase activity, whereas inactive PKC-α mutant (dnPKC-α) blocks recovery of F₀F₁-ATPase activity after injury in renal proximal tubules (RPTC). This study tested whether mitochondrial PKC-α interacts with and phosphorylates F₀F₁-ATPase. Wild-type PKC-α (wtPKC-α) and dnPKC-α were overexpressed in RPTC to increase their mitochondrial levels, and RPTC were exposed to oxidant or hypoxia. Mitochondrial levels of the γ-subunit, but not the α- and β-subunits, were decreased by injury, an event associated with 54% inhibition of F₀F₁-ATPase activity. Overexpressing wtPKC-α blocked decreases in γ-subunit levels, maintained F₀F₁-ATPase activity, and improved ATP levels after injury. Deletion of PKC-α decreased levels of α-, β-, and γ-subunits, decreased F₀F₁-ATPase activity, and hindered the recovery of ATP content after RPTC injury. Mitochondrial PKC-α co-immunoprecipitated with α-, β-, and γ-subunits of F₀F₁-ATPase. The association of PKC-α with these subunits decreased in injured RPTC overexpressing dnPKC-α. Immunocapture of F₀F₁-ATPase and immunoblotting with phospho(Ser) PKC substrate antibody identified phosphorylation of serine in the PKC consensus site on the α- or β- and γ-subunits. Overexpressing wtPKC-α increased phosphorylation and protein levels, whereas deletion of PKC-α decreased protein levels of α-, β-, and γ-subunits of F₀F₁-ATPase in RPTC. Phosphoproteomics revealed phosphorylation of Ser¹⁴⁶ on the γ subunit in response to wtPKC-α overexpression. We concluded that active PKC-α 1) prevents injury-induced decreases in levels of γ subunit of F₀F₁-ATPase, 2) interacts with α-, β-, and γ-subunits leading to increases in their phosphorylation, and 3) promotes the recovery of F₀F₁-ATPase activity and ATP content after injury in RPTC.     

Association of α-subunits with nucleotide-modified β-subunits induces asymmetry in the catalytic sites of the F1-ATPase α3β3

The photoaffinity spin-labeled ATP analog, 2-N₃-SL-adenosine triphosphate (ATP), was used to covalently modify isolated β-subunits from F₁-ATPase of the thermophilic bacterium PS3. Approximately 1.2 mol of the nucleotide analog bound to the isolated subunit in the dark. Irradiation leads to covalent incorporation of the nucleotide into the binding site. ESR spectra of the complex show a signal that is typical for protein-immobilized radicals. Addition of isolated α-subunits to the modified β-subunits results in ESR spectra with two new signals indicative of two distinctly different environments of the spin-label, e.g., two distinctly different conformations of the catalytic sites. The relative ratio of the signals is approx 2∶1 in favor of the more closed conformation. The data show for the first time that when nucleotides are bound to isolated β-subunits, binding of α-subunits induces asymmetry in the catalytic sites even in the absence of the γ-subunit. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to PDV.     

Essential Role of the ε Subunit for Reversible Chemo-Mechanical Coupling in F1-ATPase

F₁-ATPase is a rotary motor protein driven by ATP hydrolysis. Among molecular motors, F₁ exhibits unique high reversibility in chemo-mechanical coupling, synthesizing ATP from ADP and inorganic phosphate upon forcible rotor reversal. The ε subunit enhances ATP synthesis coupling efficiency to > 70% upon rotation reversal. However, the detailed mechanism has remained elusive. In this study, we performed stall-and-release experiments to elucidate how the ε subunit modulates ATP association/dissociation and hydrolysis/synthesis process kinetics and thermodynamics, key reaction steps for efficient ATP synthesis. The ε subunit significantly accelerated the rates of ATP dissociation and synthesis by two- to fivefold, whereas those of ATP binding and hydrolysis were not enhanced. Numerical analysis based on the determined kinetic parameters quantitatively reproduced previous findings of two- to fivefold coupling efficiency improvement by the ε subunit at the condition exhibiting the maximum ATP synthesis activity, a physiological role of F₁-ATPase. Furthermore, fundamentally similar results were obtained upon ε subunit C-terminal domain truncation, suggesting that the N-terminal domain is responsible for the rate enhancement.     

Differences in the products of mitochondrial protein synthesis in vivo in human and mouse cells and their potential use as markers for the mitochondrial genome in human–mouse cell hybrids

The proteins synthesized in the mitochondria of mouse and human cells grown in tissue culture were examined by electrophoresis in polyacrylamide gels. The proteins were labelled by incubating the cells in the presence of [(35)S]methionine and an inhibitor of cytoplasmic protein synthesis (emetine or cycloheximide). A detailed comparison between the labelled products of mouse and human mitochondrial protein synthesis was made possible by developing radioautograms after exposure to slab-electrophoresis gels. Patterns obtained for different cell types of the same species were extremely similar, whereas reproducible differences were observed on comparison of the profiles obtained for mouse and human cells. Four human-mouse somatic cell hybrids were examined, and in each one only components corresponding to mouse mitochondrially synthesized proteins were detected.     

Transmission of chromosomes through the eggs and pollen of triticale × wheat F1 hybrids

Summary The substitution patterns of rye chromosomes in hexaploid triticale × wheat F₂ hybrids, along with the transmission patterns of rye chromosomes through egg cells and pollen when several of the F₁ hybrids were test crossed to triticale and wheat were investigated. The data indicated that the rye chromosome transmission through both the egg and pollen was random in number and in composition. The test crosses suggested that it was best to use wheat pollen for the transmission of rye chromosomes through the egg cells of the F₁ hybrids and triticale egg cells for the transmission of rye chromosomes through F₁ hybrid pollen. A deviation from random segregation in the F₂ and the transmission rate was observed for rye chromosomes 1R, 4R/7R, and 6R. The transmission rates of 1R and 6R varied depending on the direction in which the cross was made. The results also indicated that there was little or no compensation between the R- and D-genomes and that the chromosomes of these two genomes appeared to be transmitted independently of each other.     

α,β-Bidentate CrADP abolishes the negative cooperativity of yeast mitochondrial F1-ATPase

The hydrolysis of MgATP and MgITP by mitochondrial F₁-ATPase from Saccharomyces cerevisiae is competitively inhibited by α,β-CrADP, α,β,γ-CrATP and β,γ-CrATP. The apparent K₁ values of the three complexes are in the range of the half-saturating MgATP concentration. The negative cooperativity (n_H = 0.7) of MgATP hydrolysis is totally abolished by α,β-CrADP (n_H = 1.0), while it is not affected by the CrATP. It is concluded that α,β-CrADP binds exclusively at the regulatory site and that CrATP binds exclusively to the catalytic site.     

Single-molecule Study on the Temperature-sensitive Reaction of F1-ATPase with a Hybrid F1 Carrying a Single ��(E190D)

F₁-ATPase is a rotary molecular motor in which the γ-subunit rotates against the α₃β₃ cylinder. The unitary γ-rotation is a 120° step comprising 80 and 40° substeps, each of these initiated by ATP binding and ADP release and by ATP hydrolysis and inorganic phosphate release, respectively. In our previous study on γ-rotation at low temperatures, a highly temperature-sensitive (TS) reaction step of F₁-ATPase from thermophilic Bacillus PS3 was found below 9 °C as an intervening pause before the 80° substep at the same angle for ATP binding and ADP release. However, it remains unclear as to which reaction step the TS reaction corresponds. In this study, we found that the mutant F₁(βE190D) from thermophilic Bacillus PS3 showed a clear pause of the TS reaction below 18 °C. In an attempt to identify the catalytic state of the TS reaction, the rotation of the hybrid F₁, carrying a single copy of βE190D, was observed at 18 °C. The hybrid F₁ showed a pause of the TS reaction at the same angle as for the ATP binding of the incorporated βE190D, although kinetic analysis revealed that the TS reaction is not the ATP binding step. These findings suggest that the TS reaction is a structural rearrangement of β before or after ATP binding.F₁-ATPase (F₁)² is an ATP-driven rotary motor protein. The subunit composition of the bacterial F₁-ATPase is α₃β₃γδϵ, and the minimum complex of F₁-ATPase as a rotary motor is α₃β₃γ subcomplex. This motor protein forms the F_oF₁-ATP synthase complex by binding to another rotary motor, namely, F_o, which is driven by the proton flux resulting from the proton motive force across the membranes (1–4). Under physiological conditions, where the proton motive force is sufficiently large, F_o forcibly rotates F₁-ATPase in the reverse direction of F₁-ATPase, leading the reverse reaction of ATP hydrolysis, i.e. ATP synthesis from ADP and inorganic phosphate (P_i). When the proton motive force diminishes or F₁ is isolated from F_o, F₁-ATPase hydrolyzes ATP to rotate the γ-subunit against the α₃β₃ stator ring in the counterclockwise direction as viewed from the F_o side (5). The catalytic sites are located at the interface of the α- and β-subunits, predominantly on the β-subunit (6). Each β-subunit carries out a single turnover of ATP hydrolysis during the γ-rotation of 360° following the common catalytic reaction pathway, whereas they are 120° different in the catalytic phase. In this manner, the three β-subunits undergo different reaction steps of ATP hydrolysis upon each rotational step. The rotary motion of the γ-subunit has been demonstrated by biochemical (7) and spectroscopic methods (8) and directly proved in single-molecule observation studies (5).Since the establishment of the single-molecule rotation assay, the chemomechanical coupling scheme of F₁ has been studied extensively by resolving the rotation into discrete steps. The stepping rotation was first observed under an ATP-limiting condition where F₁ makes discrete 120° steps upon ATP binding (9). Then, high speed imaging of the rotation with a small probe of low friction was performed, which revealed that the 120° step comprises 80 and 40° substeps, each initiated by ATP binding, and two unknown consecutive reactions, respectively (10). This finding necessitated the identification of the two reactions that trigger the 40° substep. Hence, the rotation assay was performed using a mutant, namely F₁(βE190D), and a slowly hydrolyzed ATP analog, namely ATPγS (11). Glutamate 190 of the β-subunit of F₁, derived from thermophilic Bacillus PS3 and the corresponding glutamates from other F₁-ATPases (Glu-181 of F₁ from Escherichia coli and Glu-188 of F₁ from bovine mitochondria), has been identified as one of the most critical catalytic residues for ATP hydrolysis (6, 12–15). When this glutamate was substituted with aspartic acid, which has a shorter side chain than that of glutamate, the ATP cleavage step of F₁ was drastically slowed. In the rotation assay, this mutant showed a distinct long pause before the 40° substep. ATPγS also caused a long pause before the 40° substep. These observations established that the 40° substep is initiated by hydrolysis. Accordingly, the pause angles before the 80 and 40° substeps are referred to as to the binding angle and the catalytic angle, respectively. Then, the rotation assay was performed in the presence of a high amount of P_i in the solution. It was shown that P_i rebinding caused the long pause at the catalytic angle, suggesting that P_i is released before the 40° substep (16).However, the reaction scheme of F₁ cannot be established by simply assigning each reaction step to either the binding angle or the catalytic angle, because each reaction step must be assigned to one of the three binding or catalytic angles when considering the 360° cyclic reaction scheme of each β-subunit. Direct information about the timing of ADP release was obtained by simultaneous imaging of fluorescently labeled nucleotides and γ rotation, which showed that each β retains ADP until the γ rotates 240° after binding of the nucleotide as ATP and releases ADP between 240 and 320° (16, 17). Another powerful approach is the use of a hybrid F₁ carrying a mutant β that causes a characteristic pause during the rotation. In a previous study, the hybrid F₁ carrying a single copy of β(E190D), α₃β₂β(E190D)γ, showed a distinct pause caused by the slow hydrolysis of β(E190D) at +200° from the ATP binding angle of the mutant β (18). From this observation, it was confirmed that each β executes the chemical cleavage of the bound ATP at +200° from the angle where the ATP binds to β. The asymmetric feature of the pause of the hybrid F₁ was also utilized in other experiments as a marker in the rotational trajectory to correlate the rotational angle and the conformational state of β (19) or to determine the state of F₁ in the crystal structures as the pausing state at catalytic angle (20).Recently, we have found a new reaction intermediate of F₁ rotation as a clear intervening pause before the 80° substep in the rotation assay below 9 °C (21). Furuike et al. (22) also observed the TS reaction in a high speed imaging experiment. The rate constant of this reaction was remarkably sensitive to temperature, giving a Q₁₀ factor around 19. When ADP was added to solution, the pause before the 80° substep was prolonged, whereas the solution P_i caused a longer pause before the 40° substep (21). Although this result can be explained by assuming that the temperature-sensitive (TS) reaction is ADP release, it was not decisive for the identification of the TS reaction.In this study, we found that the mutant F₁(βE190D) also exhibits the distinct pause of the TS reaction but at a higher temperature than for the wild-type F₁, i.e. at 18 °C. This feature was advantageous in identifying the angle position of the TS reaction in the catalytic cycle for each β-subunit coupled with the 360° rotation. Taking advantage of the feature of the hybrid F₁, we analyzed the rotational behavior of the hybrid F₁ at 18 °C in order to assign the angle position of the TS reaction in the catalytic cycle of the 360° rotation, and we have shown that the TS reaction is not directly involved in the ADP release but in some conformational rearrangement before or after ATP binding step.     

A message emerging from development: the repression of mitochondrial β-F1-ATPase expression in cancer

Mitochondrial research has experienced a considerable boost during the last decade because organelle malfunctioning is in the genesis and/or progression of a vast array of human pathologies including cancer. The renaissance of mitochondria in the cancer field has been promoted by two main facts: (1) the molecular and functional integration of mitochondrial bioenergetics with the execution of cell death and (2) the implementation of ¹⁸FDG-PET for imaging and staging of tumors in clinical practice. The latter, represents the bed-side translational development of the metabolic hallmark that describes the bioenergetic phenotype of most cancer cells as originally predicted at the beginning of previous century by Otto Warburg. In this minireview we will briefly summarize how the study of energy metabolism during liver development forced our encounter with Warburg’s postulates and prompted us to study the mechanisms that regulate the biogenesis of mitochondria in the cancer cell.     

Chromosome elimination and chromosome pairing in tetraploid hybrids of Hordeum vulgare × H. bulbosum

Summary The C₀ tetraploid counterparts of diploid hybrids of Hordeum vulgare × H. bulbosum were meiotically analysed, and were found to be chromosomally less stable than the same genotypes had been as diploids. The 14 bulbosum chromosomes present in the tetraploid cytotypes were probably eliminated as pairs rather than randomly or one genome at the time. Development of the vulgare and bulbosum genomes was asynchronous in some hybrids, the bulbosum chromosomes appearing less advanced than the vulgare chromosomes in the same cell. This appeared to reduce pairing between bulbosum homologues and also suppressed homoeologous pairing.