Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976     

Thidiazuron substitution for chilling requirement in three apple cultivars

Thidiazuron [(TDZ)N-phenyl-N-1,2,3-thidiazol-5-ylurea] at 750 M was applied to buds of apple trees to determine if it could substitute for the chilling requirement to induce bud break. Shoots of cv. Anna (low chill), Delicious cv. Redchief (medium chill), and Northern Spy (high chill) were untreated, treated with TDZ prior to chilling (before-chill), and treated with TDZ at various intervals after the accumulation of specific amounts of chilling (after-chill). Shoots were stored in a cold room at 4°C. TDZ applied prior to chilling reduced the chill unit (CU) requirement (1 CU = 1 h at 4°C) for the promotion of bud break on 1-year-old shoots of Anna and Northern Spy and 1- and 2-year-old wood of Delicious. TDZ applied after-chill promoted bud break only for Anna and buds on 2-year-old wood of Delicious. While accumulating CUs, untreated buds or buds treated with TDZ on 1-year Delicious and Northern Spy did not respond to the cold treatment even after 1848 h of CU accumulation. For all three cultivars, TDZ treatment was more effective in promoting bud break when applied before the initiation of chilling.The use of a company or product name does not constitute an endorsement by USDA or the University of Maryland nor imply approval to the exclusion of other suitable products.     

A simple brassinolide analogue 2α, 3α-dihydroxy-17β-(3-methyl-butyryloxy)-7-oxa-B-homo-5α-androstan-6-one which induces bean second internode splitting

A new synthetic brassinolide analogue, 2,3-dihydroxy-17-(3-methylbutyryloxy)-7-oxa-B-homo-5-androstan-6-one (11), has been shown to exhibit typical brassinolide activity characterised by elongation, swelling, twisting and splitting of the bean second internode. It was prepared from the known lactone 2,3,17-trihydroxy-7-oxa-B-homo-5-androstan-6-one (4) which was transformed to an isopropylidenedioxy derivative. After protection of the 2- and 3-hydroxy groups it yielded the 2,3-isopropylidenedioxy-17-(3-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one (7) on treating with 3-methylbutyryl chloride in pyridine. The analogue with a 2-methylbutyric moiety (10, 2,3-dihydroxy-17-(2-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one) in position 17 stimulated only elongation and swelling of the bean second internode. However, in this bioassay 100 times more 10 or 11 compared to 24-epibrassinolide is required to obtain the same effects. Analogues with -oriented hydroxyl groups at C-2 and C-3 (14,15), a 6-ketone (17,18) or 6-oxa-7-oxo-lactone system (12,13) in ring B lack the typical brassinolide activity. In addition, the active brassinosteroids applied to the second internode stimulated a similar, but 30% lower elongation of the first internode. From data presented here we conclude that the presence of two hydroxy groups in the positions 22 and 23 of the brassinolide side chain, which are considered as a key structural requirement, is not absolutely necessary for a compound to exhibit typical brassinosteroid activity. Nevertheless, these compounds have generally 2–10 times lower activity than that having 22,23-vicinal diol in the side chain.     

Evidence of downstream migration of Sakhalin taimen, <Emphasis Type="Italic">Hucho perryi</Emphasis>, as revealed by Sr : Ca ratios of otolith

The migratory history of Sakhalin taimen, Hucho perryi, was examined in terms of strontium (Sr) and calcium (Ca) uptake in the otolith by using wavelength dispersive X-ray spectrometry on an electron microprobe. Otolioth Sr:Ca ratios of freshwater-reared samples remained consistently at low levels throughout the otolith. The Sr:Ca ratios of samples from Lake Aynskoye of Sakhalin Island showed a low value from the core up to a point of 700–2140µm. Thereafter, the ratios increased sharply and remained at higher levels up to the outermost regions. The difference in Sr:Ca ratio might be the result of the presence of individuals that underwent seawater and freshwater life history phases, probably reflecting the ambient salinity or the seawater–freshwater gradient in Sr concentration. Otolith Sr:Ca ratio analysis revealed downstream migration history in H. perryi.     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

Structure of the yolksac epithelium and gills in the early developmental stages of rainbow trout (Salmo gairdneri) maintained in different ambient salinities

Synopsis Small numbers of mitochondria-rich (chloride) cells were found in the yolksac epithelium of rainbow trout (Salmo gairdneri) embryos just before hatching and in eleuthero-embryos up to 14 days after hatching. This suggests that the yolksac epithelium may play a limited ionoregulatory role in this species.Mitochondria-rich cells were also present in small numbers in the branchial epithelium of embryos just before hatching and in increasing numbers in eleutheroembryos during the first two weeks after hatching. The cells in the branchial epithelium showed marked variations in appearance, particularly in the fine structure of the tubular (smooth) endoplasmic reticulum. Few of the mitochondria-rich cells examined here had the pitted apex which is characteristic of homologous cells in other species.There appeared to be no differences in the numbers or appearance of mitochondria-rich cells in embryos and eleutheroembryos reared in different ambient salinities (distilled water, 110/00 and 130/00 sea water), possibly indicating that the genesis of the ionoregulatory function of the gills has not occurred at that interval of development.     

Sialidase of swine influenza A viruses: variation of the recognition specificities for sialyl linkages and for the molecular species of sialic acid with the year of isolation

The sialidase of swine influenza A viruses of N1 and N2 subtypes, isolated from 1930 to 1992, was studied for substrate specificity with ganglio-series, lacto-series type II and GM3 gangliosides containing Neu5Ac2-3Gal, Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. All viral sialidases tested showed that the activity for hydrolysing substrates with Neu5Ac2-3Gal was higher than the activities with Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. When GM1b, GM3 and sialylparagloboside were used as substrates, the earliest strain (A/Wisconsin/15/30 H1N1, isolated in 1930) showed the activity ratio of Neu5Ac2-6Gal to Neu5Ac2-3Gal to be 0.13:0.2, and the ratio Neu5Gc2-3Gal/Neu5Ac2-3Gal to be 0.19:0.37, while those strains isolated from 1978 to 1992 exhibited ratios of 0.29:0.58 for Neu5Ac2-6Gal/Neu5Ac2-3Gal and 0.51:0.76 for Neu5Gc2-3Gal/Neu5Ac2-3Gal. The above results indicate that the substrate specificities of sialidases from swine influenza A viruses towards sialyl linkages and the molecular species of sialic acid are related to the year of isolation, i.e. strains isolated after 1978 exhibited higher activity towards Neu5Ac2-6Gal and Neu5Gc2-3Gal linkages when compared with strains isolated in an earlier year, 1930.Abbreviation Neu5Ac 5-N-acetylneuraminic acid - Neu5Gc 5-N-glycolyneuraminic acid - Gal d-galactose - Glc d-glucose - Cer Ceramide - II³(Neu5Ac)Lac Neu5Ac2-3Gal1-4Glc - GM3(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-4Glc1-Cer - GM3(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-4Glc1-Cer - GM1b(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-3GalNac1-4Gal1-4Glc1-Cer - GMlb(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-3GalNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Ac)nLc4Cer Neu5Ac2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Gc)nLc4Cer Neu5Gc2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV⁶(Neu5Ac)nLc4Cer Neu5Ac2-6Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - TDC taurodeoxycholate.     

Nucleotide regulation of vasoactive intestinal peptide binding to bovine thyroid plasma membranes

The specific binding of vasoactive intestinal peptide (VIP) to bovine thyroid plasma membranes is inhibited by guanine nucleotides. Guanosine 5-triphosphate (GTP) and the non-hydrolyzable GTP analogs guanosine 5-,-imidotriphosphate (Gpp(NH)p) and guanosine 5-O-(3-thiotriphosphate) (GTP--S) inhibited markedly the binding of VIP to its receptors. This inhibition was higher with GTP than with Gpp(NH)p and GTP--S and was due to an increase of the rate of dissociation of peptide bound to membranes. Other nucleotides did not show any effect.     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

Efficient determination of angles subtended by Cα-Hα and N-HN vectors in proteins via dipole-dipole cross-correlation§

The angle CH,NHN subtended by the internuclear vectors 13C-H and 15N-HN in doubly-labeled proteins can be determined by observing the effect of cross-correlation between the dipolar interactions on zero- and double-quantum coherences involving 13C and 15N. Two complementary 2D experiments with the appearance of 15N-HN correlation spectra yield signal intensities that depend on the rate of interconversion through cross-correlated relaxation of in-phase and doubly antiphase zero- and double-quantum coherences. The ratio of the signal intensities in the two experiments bears a simple relationship to the cross-correlation rate, and hence to the angle CH,NHN. Assuming planarity of the peptide bond, the dihedral angle (between C and C) can be determined from the knowledge of CH,NHN. The experiments are very time-effective and provide good sensitivity and excellent spectral resolution.     

Genetic analysis of gliadin-encoding genes reveals gene clusters as well as single remote genes

Summary Analysis of F₂ grains from two different crosses has revealed a complex organization of the family of gliadin-coding genes located on chromosomes of the first homoeological group in hexaploid wheat. Chromosome 1A of variety Bezenchukskaya 98 was found to carry at least five gliadin-coding genes of which three genes form a cluster controlling the synthesis of the GLD1A1 block. Two additional genes are located on the both sides of this cluster and recombine with it at frequencies of 5±1.3% and 13±2.9%. Gliadinencoding genes recombining with the main clusters were also found on chromosomes 1B and 1A in the Bezenchukskaya 98 and Saratovskaya 210 varieties, respectively. In Chinese Spring, widely used in genetic studies, we discovered a recombination between genes located on chromosome 1A and controlling the synthesis of - and -gliadins. Varieties and biotypes of one variety may differ by the presence or absence of such selfish (not included in clusters) gliadin components. The similarity of organization of prolamine-coding genes on chromosomes in different cereals is considered.     

Heterochromatin variation in Cryptobothrus chrysophorus

The endemic grasshopper Cryptobothrus chrysophorus is widely distributed throughout S.E. Australia and its populations display an extensive and spectacular pattern of autosomal variation. While the standard telocentric complement of three long (L1–3), six medium (M4–9) and two short (S10–11) autosome pairs is present throughout most of its range, two quite distinct chromosome races can be defined within this species. Populations in the northern part of its distribution (northern N.S.W. and southern Queensland-northern race) are differentiated from the remainder (southern race) by fixed blocks of distal heterochromatin on autosomes M4, 5, 6, 8 and 9 and by differences in the character of the megameric M7 chromosome. Additionally, while many populations in both races show a polymorphic system of supernumerary segments on the two smallest autosomes (S10–11), that found in the northern race is both more variable and more complex. On the other hand all the populations of the southern race we have examined are polymorphic for a series of centric shifts which convert telocentrics into acro- or meta-centrics. These occur more commonly in the megameric M7 and the two smallest autosomes (S10–11) although in one population (Forbes Creek, N.S.W.) at least 12 different shifts involving 8 of the autosomes (L3, M4, 5, 6, 7, 8, 9 and S10) are known. By contrast, in the northern erace only the small autosomes (S10–11) show centric shifts. These several floating and fixed variants thus involve all chromosomes of the standard set other than the two largest autosomes (L1–2) and the X-chromosome, which appear to be invariate. Finally, morphologically distinct supernumerary (B) chromosomes, intermediate in size between the standard S10 and the M9 elements, are found in both races but are especially common in Tasmania, the most southerly point of the species range. These B-chromosomes are partly heterochromatic and partly euchromatic so that they too add to the considerable heterochromatin variation in this species.     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Variation in the karyotype of three cultivars of Narcissus tazetta L. (Amaryllidaceae)

Three cultivated varieties of Narcissus tazetta, Grand Soleil d'Or, Chinese Sacred Lily and Cypri, are triploid (2n = 3x+30) with the basic number 10. Grand Soleil d'Or has three homomorphic sets, each comprising 2 long submetacentrics, 4 long acrocentrics and 4 short acrocentrics. Karyotypes of the other two varieties are heteromorphic. Both possess one telocentric satellited chromosome. In addition, Cypri shows translocation between two chromosomes belonging to the seventh and eighth triplets. The number of secondary constrictions varies between 3 (Chinese Sacred Lily) and 4 (Grand Soleil d'Or and Cypri) which is also the number of nucleoli observed in the respective varieties.     

Contrasting leaf and ‘ecosystem’ CO2 and H2O exchange in Avena fatua monoculture: Growth at ambient and elevated CO2

Elevated CO₂ (ambient + 35 Pa) increased shoot dry mass production in Avena fatua by 68% at maturity. This increase in shoot biomass was paralleled by an 81% increase in average net CO₂ uptake (A) per unit of leaf area and a 65% increase in average A at the ecosystem level per unit of ground area. Elevated CO₂ also increased ecosystem A per unit of biomass. However, the products of total leaf area and light-saturated leaf A divided by the ground surface area over time appeared to lie on a single response curve for both CO₂ treatments. The approximate slope of the response suggests that the integrated light saturated capacity for leaf photosynthesis is 10-fold greater than the ecosystem rate. Ecosystem respiration (night) per unit of ground area, which includes soil and plant respiration, ranged from-20 (at day 19) to-18 (at day 40) mol m^-2 s^-1 for both elevated and ambient CO₂ Avena. Ecosystem below-ground respiration at the time of seedling emergence was -10 mol m^-2 s^-1, while that occuring after shoot removal at the termination of the experiment ranged from -5 to-6 mol m^-2 s^-1. Hence, no significant differences between elevated and ambient CO₂ treatments were found in any respiration measure on a ground area basis, though ecosystem respiration on a shoot biomass basis was clearly reduced by elevated CO₂. Significant differences existed between leaf and ecosystem water flux. In general, leaf transpiration (E) decreased over the course of the experiment, possibly in response to leaf aging, while ecosystem rates of evapotranspiration (ET) remained constant, probably because falling leaf rates were offset by an increasing total leaf biomass. Transpiration was lower in plants grown at elevated CO₂, though variation was high because of variability in leaf age and ambient light conditions and differences were not significant. In contrast, ecosystem evapotranspiration (ET) was significantly decreased by elevated CO₂ on 5 out of 8 measurement dates. Photosynthetic water use efficiencies (A/E at the leaf level, A/ET at the ecosystem level) were increased by elevated CO₂. Increases were due to both increased A at leaf and ecosystem level and decreased leaf E and ecosystem ET.     

Oligodendrocytes in the normal and chronically de-afferented lateral geniculate body of the monkey

Summary Electron microscopical examination of the norma and de-afferented laterall geniculate body of the monkey following paraformaldehyde-glutaraldehyde vascular perfusion revealed distinctive morphological features of different types of oligodendrocyte. These cells were normally situated as perineuronal satellites or in relation to axons and capillaries. A wide range of nuclear and cytoplasmic densities were displayed by both satellite and interfascicular oligodendrocytes. The following distinctive features for the identification of ligodendrocytes were utilised: the presence of large quantities of free ribosomes and ribosomal rosettes, microtubular profiles, dense marginal aggregation of nuclear chromatin together with light patches and numerous nuclear pores; but the absence of broad cytoplasmic processes, glycogen and gliofibrils. Circumferential perinuclear organization of the cytoplasmic organelles was typical of oligodendrocytes. Particular attention was paid to perineuronal satellite cells in view of the known transneuronal atrophy in the de-afferented geniculate body. Some cells having a nuclear pattern of oligodendrocytes but showing hyalinisation of perikaryon were seen in de-afferented laminae. A notable feature was the presence of variegated osmiophilic bodies in the perikaryon of oligodendrocytes also situated in the de-afferented laminae. A cell type combining the features of oligodendrocytes and astrocytes was classified as intermediate neuroglia.Fellow of the Alexander von Humboldt Foundation, on Sabbatical leave from J. Nehru Medical College Aligarh, India.Recipient of the Deutsche Forschungsgemeinschaft Grant No. G./28/15.     

ATP Synthases in the Year 2000: Defining the Different Levels of Mechanism and Getting a Grip on Each

ATP synthases are unusually complex molecules, which fractionate most readily into two major units, one a water soluble unit called F₁ and the other a detergent soluble unit called F₀. In almost all known species the F₁ unit consists of 5 subunit types in the stoichiometric ratio ₃₃ while the F₀ unit contains 3 subunit types (a, b, and c) in E. coli, and at least 10 subunit types (a, b, c, and others) in higher animals. It is now believed by many investigators that during the synthesis of ATP, protons derived from an electrochemical gradient generated by an electron transport chain are directed through the F₀ unit in such a way as to drive the rotation of the single subunit, which extends from an oligomeric ring of at least 10 c subunits in F₀ through the center of F₁. It is further believed by many that the rotating subunit, by interacting sequentially with the 3 pairs of F₁ (360° cycle) in the presence of ADP, P_i, and Mg⁺⁺, brings about via power strokes conformational/binding changes in these subunits that promote the synthesis of ATP and its release on each pair. In support of these views, studies in several laboratories either suggest or demonstrate that F₀ consists in part of a proton gradient driven motor while F₁ consists of an ATP hydrolysis driven motor, and that the subunit does rotate during F₁ function. Therefore, current implications are that during ATP synthesis the former motor drives the latter in reverse via the subunit. This would suggest that the process of understanding the mechanism of ATP synthases can be subdivided into three major levels, which include elucidating those chemical and/or biophysical events involved in (1) inducing rotation of the subunit, (2) coupling rotation of this subunit to conformational/binding changes in each of the 3 pairs, and (3) forming ATP and water (from ADP, P_i, and Mg⁺⁺) and then releasing these products from each of the 3 catalytic sites. Significantly, it is at the final level of mechanism where the bond breaking/making events of ATP synthesis occur in the transition state, with the former two levels of mechanism setting the stage for this critical payoff event. Nevertheless, in order to get a better grip in this new century on how ATP synthases make ATP and then release it, we must take on the difficult challenge of elucidating each of the three levels of mechanism.