ATP synthases—Structure of the F1-moiety and its relationship to function and mechanism

A great deal of progress has been made in understanding both the structure and the mechanism of F₁-ATPase. The primary structure is now fully known for at least five species. Sequence comparison between chloroplast, photobacteria, aerobic bacteria, and mitochondrial representatives allow us to infer more general functional relationships and evolutionary trends. Although the F₁ moiety is the most studied segment of the H⁺-ATPase complex, there is not a full understanding of the mechanism and regulation of its hydrolytic activity. The subunit is now known to contain one and probably two nucleotide binding domains, one of which is believed to be a catalytic site. Recently, two similar models have been proposed to attempt to describe the active part of the subunits. These models are mainly an attempt to use the structure of adenylate kinase to represent a more general working model for nucleotide binding phosphotransferases. Labelling experiments seem to indicate that several critical residues outside the region described by the adenylate kinase part of this model are also actively involved in the ATPase activity. New models will have to be introduced to include these regions. Finally, it seems that a consensus has been reached with regard to a broad acceptance of the asymmetric structure of the F₁-moiety. In addition, recent experimental evidence points toward the presence of nonequivalent subunits to describe the functional activity of the F₁-ATPase. A summary diagram of the conformational and binding states of the enzyme including the nonequivalent subunit is presented. Additional research is essential to establish the role of the minor subunits—and of the asymmetry they introduce in F₁—on the physiological function of the enzyme.     

Inheritance of the dwarf plant type in blackgram (Vigna mungo (L.) Hepper)

Summary The inheritance of the dwarf plant type was studied in blackgram (V. mungo (L.) Hepper). Type 9 has erect plant type with normal internode length. The mutant line, EMSD has reduced internode length. The F₁, F₂ and F₃ generations of a cross between Type 9 and EMSD and its reciprocal were studied. The extreme dwarf plant type appeared to be governed by a single recessive gene, dw ₁ dw ₁ with no cytoplasmic effect.Part of Ph.D. Thesis submitted by the first author     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

Immunochemistry of membrane F1-Adenosine triphosphatase fromMicrococcus lysodeikticus. Role of the alpha subunit

The membrane-bound ATPase activity from two substrains ofMicrococcus lysodeikticus, designated as A and B, was inhibited by antibodies raised against the two forms of purified F₁-ATPase. Form B of the enzyme, which behaved as a poorer immunogen than form A, also showed less reactivity as an antigen, independent of the physical state of the F₁-ATPase form. Antibodies were raised against the two major subunits ( and ) isolated fromM. lysodeikticus F₁-ATPase form A, which was the most stable form of the enzyme. Anti-(-subunit) serum strongly inhibited the ATPase activity of membrane-bound ATPase but showed little inhibition of the purified, soluble F₁-ATPase. The anti-(-subunit) serum inhibited the soluble F₁-ATPase, but to a lesser extent than the membrane-bound enzyme. In any event, the effect of anti- antibodies on the membrane-bound ATPase was smaller than that of anti- antibodies. It was postulated that the subunit ofM. lysodeikticus F₁-ATPase plays an essential and regulatory role in the expression of the immunochemical properties of the protein.     

Theoretical stability diagram of solvent-containing black lipid films

Recently, we have developed an analytical, semi-microscopic theory for the macroscopic behavior of a solvent-containing black lipid film subjected to an electric cross film voltage, . Here we employ the theoretical expressions derived for the disjoining pressure, _D, the film elasticity, _F, and the film tension, _F, to construct the stability diagram of the film, in the _D-. Depending on its state (_D, ), the film is stable or is prone to squeezing or bending deformations. For a monooleate film we show how the destruction of the plane film due to a periodic thickness fluctuation (squeezing) is facilitated by two mechanisms: i) lowering of _D at fixed ; ii) lowering of at fixed _D, provided that the film is in a stable state characterized by _D<–7.03×10³ dyne/cm² and >0 mV. Bending of a low tension film (single interface tension _s 0.025 dyne/cm¹) can be achieved only for >170 mV and _D > –8.7 × 10⁴ dyne/cm². Finally, we demonstrate the existence of a marginal state ( _D ⁰ , ⁰) where the film is predicted to exhibit strong fluctuations both in the squeezing and in the bending mode.     

Identification of subunits required for the catalytic activity of the F1-ATPase

F₁() complexes containing equimolar ratios of the and subunits have been shown to function as active ATPases, whereas individually isolated and subunits show no real ATPase activity. These results indicate that the single-copy subunits are not required for F₁-ATPase activity. The minimal F₁()-core complexes exhibit, however, lower rates and some different properties from those of their parent whole F₁ or ₃₃ complexes. It is therefore concluded that for obtaining a full spectrum of the characteristic functional properties of an F₁-ATPase the presence of the F₁- subunit is also required. The implications of these findings on the subunit location of both catalytic and noncatalytic nucleotide binding sites is discussed.     

Endothelin-1 and insulin induce cellular inactivation of protein kinase FA/glycogen synthase kinase-3α in a common signaling pathway

In this study, we investigate the effects of endothelin-1 (ET-1) and insulin on the cellular activity of protein kinase F_A/glycogen synthase kinase-3 (kinase F_A/GSK-3) in rat adipocytes. The cellular activity of kinase F_A/GSK-3 is inhibited to 50% of control within 30 min when cells are treated with 1 nM ET-1 at 37°C; in addition, significant inhibition to 60% of control is observed at as low as 1 pM ET-1. Conversely, ET-1 at concentrations up to 1 nM has no direct effect on purified kinase F_A/GSK-3 in vitro. Immunoblotting analysis further reveals that the protein level of this kinase is not significantly changed when treated with 1 nM ET-1 for 30 min. Similar to ET-1, insulin as low as 10 nM can also induce inactivation of kinase F_A/GSK-3 to 50% of control in adipocytes when processed under identical conditions. Most importantly, when treated with both insulin and ET-1, the activity of kinase F_A/GSK-3 can be decreased only to 50% of control. Taken together, the results provide initial evidence that ET-1 and insulin may regulate this important multisubstrate/multifunctional protein kinase in a common signaling pathway in cells.     

Structure and function of H+-ATPase

(1) Extensive studies on proton-translocating ATPase (H⁺-ATPase) revealed that H⁺-ATPase is an energy transforming device universally distributed in membranes of almost all kinds of cells. (2) Crystallization of the catalytic portion (F₁) of H⁺-ATPase showed that F₁ is a hexagonal molecule with a central hole. The diameter of F₁ is about 90 Å and its molecular weight is about 380,000. (3) Use of thermophilic F₁ permits the complete reconstitution of F₁ from its five subunits (, , , , and ) and demonstration of the gate function of the -complex, the catalytic function of (supported by and ), and the H⁺-translocating functions of all five subunits. (4) Studies using purified thermostable F₀ showed that F₀ is an H⁺-channel portion of H⁺-ATPase. The direct measurement of H⁺-flux through F₀, sequencing of DCCD-binding protein, and isolation of F₁-binding protein are described. (5) The subunit stoichiometry of F₁ may be ₃₃. (6) Reconstitution of stable H⁺-ATPase-liposomes revealed that ATP is directly synthesized by the flow of H⁺ driven by an electrochemical potential gradient and that H⁺ is translocated by ATP hydrolysis. This rules out functions for all the hypothetical components that do not belong to H⁺-ATPase in H⁺-driven ATP synthesis. The roles of conformation change and other phenomena in ATP synthesis are also discussed.     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.     

Interaction of high-affinity nucleotide binding sites in mitochondrial ATP synthesis and hydrolysis

The present study contributes to the problem of the dynamic structure of mitochondrial F1-ATPase and the functional interrelation of so-called tight nucleotide binding sites. Nucleotide analogs are used as a tool to differentiate two distinct functional states of the membrane-bound enzyme, proposed to reflect corresponding conformational states; they reveal F1-ATPase as a dual-state enzyme: ATP-synthetase, and ATP-hydrolase. The analogs used are 3-naphthoyl esters of AD(T)P, and 2(3)-O-trinitrophenyl ethers of AD(T)P. Both types of analogs act inversely to each other with respect to their relative effects on oxidative phosphorylation and on ATPase in submitochondrial vesicles. The respective ratios ofK _i versus both processes are 250/1 compared to 1/170. It is also shown that in the presence of the inhibitory 3-esters oxidative phosphorylation deviates from linear kinetics and that these inhibitors induce a lag time of oxidative phosphorylation depending on the initial pattern of nucleotides available to energized submitochondrial vesicles. The duration of the lag time coincides with the time course of displacement of the analog from a tight binding site. The conclusions of the study are: (a) the catalytic sites of F₁-ATP-synthetase are not operating independently from each other; they rather interact in a cooperative manner; (b) F₁-ATPase as a dual-state enzyme exhibits highly selective responses to tight binding of nucleotides or analogs in its energized (membrane-bound) state versus its nonenergized state, respectively.Abbreviations used: N-AD(T)P, 3-O-naphthoyl(1)-AD(T)P; DMAN-AD(T)P, 3-O-(5-dimethylaminonaphthoyl(1))-AD(T)P, also termed F-AD(T)P in previous papers because of its fluorescence; TNP-AD(T)P, 2(3)-O-(2,4,6-trinitrophenyl)-AD(T)P; FCCP,p-trifluoromethoxycarbonylcyanide phenylhydrazone.     

Photosynthetic ATPases: purification,properties, subunit isolation and function

Photosynthetic coupling factor ATPases (F₁-ATPases) generally censist of five subunits named , , , and in order of decreasing apparent molecular weight. The isolated enzyme has a molecular weight of between 390,000 to 400,000, with the five subunits probably occurring in a 3:3:1:1:1 ratio. Some photosynthetic F₁ ATPases are inactive as isolated and require treatment with protease, heat or detergent in order to elicit ATPase activity. This activity is sensitive to inhibition by free divalent cations and appears to be more specific for Ca²⁺ vs. Mg²⁺ as the metal ion substrate chelate. This preference for Ca²⁺ can be explained by the higher inhibition constant for inhibition of ATPase activity by free Ca²⁺. Methods for the assay of a Mg-dependent ATPase activity have recently been described. These depend on the presence of organic solvents or detergents in the reaction mixture for assay. The molecular mechanism behind the expression of either the Ca- or Mg-ATPase activities is unknown. F₁-ATPases function to couple proton efflux from thylakoid membranes or chromatophores to ATP synthesis. The isolated enzyme may thus also be assayed for the reconstitution of coupling activity to membranes depleted of coupling factor 1.The functions of the five subunits in the complex have been deduced from the results of chemical modification and reconstitution studies. The subunit is required for the functional binding of the F₁ to the F₀. The active site is probably contained in the (and ) subunit(s). The proposed functions for the and subunits are, however, still matters of controversy. Coupling factors from a wide variety of species including bacteria, algae, C₃ and C₄ plants, appear to be immunologically related. The subunits are the most strongly related, although the and subunits also show significant immunological cross-reactivity. DNA sequence analyses of the genes for the subunit of CF₁ have indicated that the primary sequence of this polypeptide is highly conserved. The genes for the polypeptides of CF₁ appear to be located in two cellular compartments. The , and subunits are coded for on chloroplast DNA, whereas the and subunits are probably nuclear encoded. Experiments involving protein synthesis by isolated chloroplasts or protein synthesis in the presence of inhibitors specific for one or the other set of ribosomes in the cell suggest the existence of pools of unassembled CF₁ subunits. These pools, if they do exist in vivo, probably make up no greater than 1% of the total CF₁ content of the cell.Abbreviations AMP-PNP adenylyl 5 imidodiphosphate - bchl bacteriochlorophyll - CF₁ chloroplast coupling factor 1 - CF₁-CF₀ the chloroplast ATP synthase complex - chl chlorophyll - CvF₁ F₁ from Chromatium vinosum - DCCD N, N-dicyclohexyl carbodiimide - EF₁ the coupling factor 1 isolated from membranes of Escherichia coli - F₀ the hydrophobic, integral membrane portion of the ATP synthase - F₁ coupling factor 1, the extrinsic membrane portion of the ATP synthase - FSBA 5-p-fluorosulfonylbenzoyladenosine - K_d dissociation constant - k_i inhibition constant - k_ii intercept inhibition constant - k_is slope inhibition constant - LS large subunit of ribulose bisphosphate carboxylase - MF₁ mitochondrial coupling factor 1 - M1F₁ F₁ from Mastigocladus laminosus - NBD-Cl 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole - PAGE polyacrylamide gel electrophoresis - RcF₁ F₁ from Rhodopseudomonas capsulata - RpF₁ F₁ from Rhodopseudomonas palustris - RrF₁ F₁ from Rhodospirillum rubrum - RsF₁ F₁ from Rhodopseudomonas sphaeroides - SDS sodium dodecyl sulfate - S1F₁ F₁ from Synechococcus lividus - SpF₁ F₁ from Spirulina platensis - TF₁ F₁ from the thermophilic bacterium, PS3 - tricine N-tris (hydroxymethyl) methyl glycine - tris tris (hydroxymethyl)-amino methane; and - Vmax maximal velocity or maximal activity     

Mutagen sensitivity and mutability in lentil

Summary Seeds of two cultivars, each of macrosperma and microsperma varietal groups of lentil were mutagenised with gamma-rays and NMU to determine their mutagen sensitivity and mutability. The increasing doeses of gamma-rays and NMU decreased germination, root and shoot length, pollen fertility and plant survival, but increased the occurrence of leaf spots. The root system was found to be more sensitive to both mutagens than the shoot. The macrosperma varietal group was more sensitive to both the mutagens than microsperma group. In microsperma group, variety Pusa-1 was more sensitive to both the mutagens than L-259, while in the macrosperma group L-1491 showed more sensitivity to the mutagens than L-1492. Radio-sensitivity corresponded positively with chemosensitivity in both varietal groups. There was a positive relationship between radio- and chemo-sensitivity of the genotypes and their mutability. The results also revealed the existence of a parallelism between radiomutability and chemo-mutability. Due to saturation in the mutational events and vigour of both diplontic and haplontic selection in the biological material, the mutation frequency either decreased or remained constant at higher doses of the mutagens.     

ATP Synthases in the Year 2000: Evolving Views about the Structures of These Remarkable Enzyme Complexes

This introductory article briefly summarizes how our views about the structural features ofATP synthases (F₀F₁) have evolved over the past 30 years and also reviews some of our currentviews in the year 2000 about the structures of these remarkably unique enzyme complexes.Suffice it to say that as we approach the end of the first year of this new millinium, we canbe conservatively confident that we have a reasonably good grasp of the overall low-resolutionstructural features of ATP synthases. Electron microscopy techniques, combined with the toolsof biochemistry, molecular biology, and immunology, have played the leading role here byidentifying the headpiece, basepiece, central stalk, side stalk, cap, and in the mitochondrialenzyme, the collar around the central stalk. We can be reasonably confident also that we havea fairly good grasp of much of the high-resolution structural features of both the F₁ moietycomprised of fives subunit types (, , , , and ) and parts of the F₀ moiety comprised ofeither three (E. coli) or at least ten (mitochondria) subunit types. This information acquiredin several different laboratories, either by X-ray crystallography or NMR spectroscopy, includesdetails about the active site and subunit relationships. Moreover, it is consistent with recentlyreported data that the F₁ moiety may be an ATP driven motor, which, during ATP synthesis,is driven in reverse by the electrochemical proton gradient generated by the electron transportchain. The real structural challenges of the future are to acquire at high resolution completeATP synthase complexes representative of different stages of the catalytic cycle during ATPsynthesis and representative also of key regulatory states.     

Weitere Untersuchungen an Nachkommen von Kürbis-Artbastarden

Zusammenfassung Aus 1950 durchgeführten Artkreuzungen zwischenCucurbita maxima Duch. und weichschaligemC. pepo L. wurden mittels Embryonenkultur F₁-Bastarde gewonnen. An Hand zweimaliger Rückkreuzung mitC. maxima und anschließender Selbstung gelang es, zum Teil wiederum mit Hilfe der Embryonenkultur, 4 verschiedene Bastardkombinationen bis in die F₄ zu verfolgen. In dieser Generation spalteten (1954 und 1955) bei Auswertung von 72 Familien mit insgesamt 733, davon 600 fertilen Pflanzen in 2 Bastardlinien resp. 3 Familien, insgesamt 4 weichschalige Pflanzen heraus. Da diese Pflanzen frei abblühten und das gewonnene Saatgut heterozygot war, mußten erneut zwei Selbstungsgenerationen aufgezogen werden, die in den Jahren 1956–58 mehrere erbreine Stämme weichschaliger Pflanzen lieferten.Nach Kurzer Einführung in die Anatomie der Samenschale und ihre verschiedenen Typen sowie in die bisherigen Ergebnisse genetischer Untersuchungen zur Frage der Testaausbildung wird an Hand der Spaltungsergebnisse beim erstmaligen Auftreten weichschaliger Pflanzen in der F₄ sowie der nach erneuter Selbstung in der F₆ erzielten Spaltungsverhältnisse aufgezeigt, daß das Merkmal weichschalig durchzwei Gene bedingt ist. Nach kurzer Besprechung von Spaltungen pachyspermer und leptospermer Pflanzen in der F₃ wird die Variabilität der Testaausbildung bis zum Auftreten weichschaliger Pflanzen beschrieben.In einem weiteren Kapitel wird die Variabilität in der Ausbildung der weichschaligen Testa der Bastardnachkommen, das Ergebnis der Selektion auf rein weichschalige Typen im Verlauf der 7. bis 11. Generation (1957–1964) und die Größenvariabilität der Samen der weichschaligen Typen im Vergleich zu den Ausgangsarten sowie einem Weltsortiment vonC. maxima erörtert. Zur Veranschaulichung dieser Verhältnisse wird die erstmalig vonF. Galton benutzte Methode der Contourellipsen herangezogen, deren Berechnung und zeichnerische Darstellung mit Hilfe der Großrechenanlage IBM 7090 erfolgte.Abschließend wurden die beobachtetenMendel-Spaltungen unter dem Gesichtspunkt der die Pollenmeiose sowie die Pollenentwicklung der F₁ charakterisierenden Störungen, ferner die Bedeutung des erzielten Ergebnisses für die Züchtung diskutiert. Es wird eine Formel für die Berechnung der Wahrscheinlichkeit mitgeteilt, mit der unter ähnlichen Bedingungen rezessive Pflanzen erwartet werden können, wenn das rezessive Merkmal durch n Faktoren bedingt ist und im Anschluß an die Artkreuzungj Rückkreuzungen mit der dominanten Art erforderlich sind. Auch auf die Bedeutung von Untersuchungen der vorliegenden Art für Fragen der Genlokalisation und Genanalyse wird hingewiesen.

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Further investigations on progeny of pumpkin species hybridsI. Seed formation, especially in progeny with soft seed coat, and the regularity in transmission of a recessive trait from one parental species to fertile progeny with characteristics of the other parental species

Summary Using embryo culture, we obtainedF ₁-hybrids from interspecific crosses realized in 1950 betweenCucurbita maxima Duch. and a form ofC. pepo L. with soft shell seed or weichschaligen Samen (a so called naked seed type). These and their descendants, the latter in part also obtained by embryo culture, were back-crossed toC. maxima, and theF ₃ selfed. In this way we obtained descendants from 4 different crosses between the two species.Studying (in 1954 and 1955) 72F ₄-families with a total of 733 (600 fertile) plants, we found 4 plants with soft shell seeds. They were descended from 3F ₄-families or from 2 crosses respectively between the above named species.Since these plants flowered in the presence of other progeny from our species crosses, their seed was heterozygous, and two generations of selfing were necessary to obtain (in 1956–1958) different true breeding families, whose plants furnished homozygous soft shell seed.The anatomy of seed coats and results of studies by other authors on the genetic control of different seed coat types are discussed briefly. Given the segregation ratios in theF ₄ of our soft shell seeds, as well as those obtained inF ₆ after renewed selfing, it is shown that the trait soft shell is controlled by two genes. After a short discussion of segregation in theF ₃ of pachyspermic and leptospermic plants, the variability of seed coat formation until the appearance of soft shell seed is described.In a special chapter we discuss the variability of the soft-shell seed character in hybrid progeny, the result of our selection experiments from the 7^th up to the 12^th generation (1957–1964), and the variability in the seed size of our types compared with that of pure species and of a collection ofC. maxima. To illustrate these relationships we used the method of contour-ellipses introduced byF. Galton. These ellipses were computed and designed by means of the computer IBM 7090.Finally we discuss Mendelian segregation of our plants in the light of abnormalities characteristic for meiosis of pollen-mother-cells and pollen development inF ₁, and the significance of the results to plant breeding. A formula is given for calculating the probability of obtaining recessive plants when the recessive trait depends onn factors, and whenj backcrosses to the dominant variety are necessary to establish a stable strain. The importance of experiments of this kind to the problem of gene localization and gene analysis is also pointed out.

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Herrn Professor Dr. Dr. Dr. h. c. O.Heinisch, geb. 23. 4. 1896, gest. 3. 5. 1966, zum Gedächtnis.     

Inheritance of alleles for Cgy1 and Gy4 storage protein genes in soybean

Summary A cultivar lacking the glycinin subunit A₅A₄B₃ (Raiden) was crossed with one lacking the -subunit of -conglycinin (Keburi). Analysis of F₂ and F₃ progeny indicated that the missing bands of the A₅A₄B₃ and the -subunit were each controlled by a recessive allele of two independently segregating genes. Gene symbols Gy ₄/gy ₄ and Cgy ₁/cgy ₁ were proposed for the genes which confer the presence or absence of the glycinin and conglycinin subunits, respectively.Cooperative research of USDA-ARS and the Indiana Agric. Exp. Stn., Purdue Univ., West Lafayette, IN 47907, USA. Indiana Agric. Exp. Stn. Journal Article 9675. Financial support from the American Soybean Association Research Foundation is gratefully acknowledged     

Formation of type II F-primes from unstable Hfrs and their recA-independent conversion to other F-prime types

Summary Four E. coli Hfr strains, representing stable (Hfr Cavalli), moderately stable (AB312) and unstable (Ra-1, Ra-2) Hfr states, were used in the isolation of a series of F plasmids. Type II Fs were found to be the most prevalent F plasmid formed from all of the Hfrs, while the percentages of tra Fs increased as the stability of the Hfr increased. Two observations suggested that F formation in unstable Hfrs like Ra-2 may proceed through a type II F precursor. First, the major F products of Ra-2 are tra ⁺ type II Fs and, second, other F types (I, II) and classes (tra ⁺, tra) from Ra-2 appeared to be deletion derivatives of a larger F progenitor. By monitoring the molecular changes that occur when the Ra-2 derived type II F pWS200 is transferred from one recA host to another, we have found that all F types and classes can be generated from pWS200 in a recA-independent manner. F sequences involved in the genetic conversions of pWS200 include the oriT locus and the directly repeated junctions of F and chromosomal DNA. A model for the formation of Fs in unstable Hfrs is postulated in which a tra ⁺ type II F primary excision product is seen to be modified, through recA-independent processes, to other F types and classes. This model differs from the current model of F formation in that independent excision events from the Hfr chromosome are not seen as the source of type I and type II Fs.These studies have also shown that the formation of tra Fs is a recA-independent process that can occur from the F and Hfr states, that -mediated deletions in pWS200 often demonstrate regional specificity in having endpoints near the ilv operon and that genetic alterations in either replication origin of pWS200 (F oriV, chromosomal oriC) stabilize the replication of this mini-Hfr cointegrate.     

Inheritance of resistance to blackmold (Alternaria alternata (Fr.) Keissler) in two interspecific crosses of tomato (Lycopersicon esculentum ×L. cheesmanii f.typicum)

Inheritance of resistance to blackmold, a disease of ripe tomato fruit caused byAlternaria alternata, was studied in two interspecific crosses. The parents, F₁ and F₂ generations of a cross between the susceptibleLycopersicon esculentum Mill. cultivar Hunt 100 and the resistantL. Cheesmanii f.typicum Riley accession LA 422, and the parents, F₁, F₂, F₃, and BC₁ P₂ generations of a cross between the susceptibleL. Esculentum cv. VF 145B-7879 and LA 422 were evaluated. The following disease evaluation traits were used: symptom rating (a symptom severity rating based on visual evaluation of lesions), diseased fruit (the number of diseased fruits divided by the total number of fruit scored), and lesion size (a function derived from the actual lesion diameter). Generation means analysis was used to determine gene action. The data of the Hunt 100 × LA 422 cross fit an additive-dominance model for all three traits. The VF 145B-7879 × LA 422 cross data best fit a model that included the additive × additive and additive × dominance interaction components for the trait diseased fruit, whereas higher-order epistatic models would have to be invoked to fit the data for the traits symptom rating and lesion size. A minimum of one gene segregated for all three traits. Broad-sense heritability estimates ranged from 0.09 to 0.16 for all three traits, indicating that selection for improved resistance to blackmold will require selection on a family performance basis.     

Autotrophic carbon sources for heterotrophic bacterioplankton in a floodplain lake of central Amazon

The relative contribution of autotrophic carbon sources (aquatic macrophytes, flooded forest, phytoplankton) for heterotrophic bacterioplankton was evaluated in a floodplain lake of the Central Amazon. Stable carbon isotopes (¹³C) were used as tracers. Values of ¹³C of different autotrophic sources were compared to those of dissolved organic carbon (DOC) and those of bacterially produced CO₂.The percentage of carbon derived from C₄ macrophytes for bacterially produced CO₂ was the highest, on average 89%. The average ¹³C value of CO₂ from bacterial respiration was –18.5 ± 3.3. Considering a fractionation of CO₂ of 3 by bacterial respiration, ¹³C value was –15.5, near C₄ macrophyte ¹³C value (–13.1).The average value of total DOC ¹³C was –26.8 ± 2.4. The percentage of C₄ macrophytes carbon for total DOC was on average 17%. Considering that bacteria consume mainly carbon from macrophytes, the dominance of C₃ plants for total DOC probably reflects a faster consumption of the former source, rather than a major contribution of the latter source.Heterotrophic bacterioplankton in the floodplain may be an important link in the aquatic food web, transferring the carbon from C₄ macrophytes to the consumers.     

Characterization of maltose biosynthesis from α-d-glucose-1-phosphate in Spinacia oleracea. L.

The de novo synthesis of maltose in spinach (Spinacia oleracea L.) was shown to be catalyzed by a maltose synthase, which converts two molecules of -d-glucose-1-phosphate (-G1P) (K_m 1.5 mmol l^-1) to maltose and 2 orthophosphate (Pi). This enzyme was purified 203-fold by fractionated ammonium sulfate precipitation and by column chromatography on Sepharose 6B. The addition of -G1P (15 mmol l^-1) to the isolation buffer is required to stabilize the enzyme activity during the extraction and purification procedure. Molecular weight determination by gel filtration yielded a value of 95,000. -Gluconolactone, ATP and Pi are competitive inhibitors toward the substrate -G1P. The maltose synthase catalyzes an exchange of the phosphate group of -G1P with [³²P] orthophosphate; this transfer reaction suggests that the synthesis of maltose occurs via a glucose-enzyme in a double displacement reaction. The physiological role of this enzyme as a starch initiator system is discussed.Abbreviations Fru fructose - Glc glucose - -G1P -d-glucose-1-phosphate - -G1P -d-glucose-1-phosphate - G6P d-glucose-6-phosphate This enzyme is tentatively called maltose synthase in this publication     

Malate regulation of Mg2+-ATPase of chloroplast coupling factor 1

The regulatory effects of malate on chloroplast Mg²⁺-ATPase were investigated and the mechanism was discussed. Malate stimulated methanol-activated membrane-bound and isolated CF₁ Mg²⁺-ATPase activity. The subunit of CF₁ may be involved in malate regulation of the enzyme function. Modification of subunit at one site of the peptide by NEM may affect malate stimulation of ATPase while at another site may have no effect. The effect of malate on the Mg²⁺-ATPase was also controlled by the Mg²⁺/ATP ratio in the reaction medium. The enhancing effect of malate on Mg²⁺-ATPase activity depended on the presence of high concentrations of Mg²⁺ in the reaction mixture. Kinetic study showed that malate raised the V_max of catalysis without affecting the K_m for Mg²⁺ ATP. The experiments imply that the stimulation of Mg²⁺-ATPase by malate is probably correlated with the P_i binding site on the enzyme. The regulation of ATPase activity by malate in chloroplasts may be relevant to its function in vivo.Abbreviations CF₁ chloroplast coupling factor 1 - CF₁ (-) and CF₁ (-) CF₁ deficient in the and subunit - MF₁ mitochondria coupling factor 1 - NEM N-ethylmaleimide - PMS phenazine methosulfate - OG n-octyl--d-glucopyranoside