Efj Vanbruggen

THE CRYSTALLINE DOMAINS IN POTATO STARCH GRANULES ARE ARRANGED IN A HELICAL FASHION

Abstract The structural basis for the physical properties of starches from different botanical sources is still poorly understood. Particularly at the level of the crystalline domains the present knowledge concerning the structure of starch is limited. This paper reports the semi-crystalline structure of potato starch granules by electron optical tomography and by cryo electron diffraction. It is concluded that the crystalline domains in the amylopectin form a continuous network of left-handed helices, which appears as a well-ordered skeleton for the starch granule. This type of super-helical lamellar structure has not been reported before, either in natural or in synthetic polymers.

TWO-DIMENSIONAL CRYSTALLIZATION OF BOVINE RHODOPSIN

Bovine rhodopsin has been clustered into two-dimensional crystals in highly purified native rod disk membranes and studied with negative staining and transmission electron microscopy. The lattice is P2(1) with dimensions of 8.3 X 7.9 nm and interaxis angles of 86 +/- 3 degrees. 110 images of ordered areas were digitized and aligned with computer-correlation methods to calculate an average image with diffraction to the fourth order. The images were computer-filtered and reconstructed to approx. 2 nm resolution. When crystals appeared they covered 20-40% of the surface of the preparation and, since rhodopsin is at least 95% of the protein, there is no doubt that the crystals were due to rhodopsin. There appear to be two rhodopsin dimers per unit cell. Each rhodopsin molecules takes up about 7.5 nm2 of membrane area and is estimated to be associated with about 12 lipids on each side of the membrane. The membrane area found for bovine rhodopsin supports the rhodopsin origin of rarely seen but more highly ordered two-dimensional crystals found in detergent-treated frog rod membranes (Corless, J.M., McCaslin, D.R. and Scott, B.L. (1982) Proc. Natl. Acad. Sci. USA 79, 1116-1120). Furthermore, the rhodopsin membrane area is close to that of bacteriorhodopsin and is consistent with a seven transmembrane helix structure proposed for rhodopsin (for references see Dratz, E.A. and Hargrave, D.A. (1983) Trends Biochem. Sci. 8, 128-131). Crystallization was accomplished by lowering the pH to 5.5 near the isoelectric point of rhodopsin, raising the salt concentration of 2 M (NH4)2SO4, adding 5% glucose and 0.02% Hibitane (Ayerst), a cationic amphipathic antiseptic that favored crystal growth.

THE QUATERNARY STRUCTURE OF 4 CRUSTACEAN 2-HEXAMERIC HEMOCYANINS - IMMUNOCORRELATION, STOICHIOMETRY, REASSEMBLY AND TOPOLOGY OF INDIVIDUAL SUBUNITS

SummaryTwo-hexameric (2×6) hemocyanins from the brachyuran crabsCancer pagurus andCallinectes sapidus, the freshwater crayfishAstacus leptodactylus and the lobsterHomarus americanus were isolated and dissociated into native subunits.The subunits of each hemocyanin were analyzed by electrophoresis and immunology. Three immunologically distinct subunit types, which were termedα,β andγ, could be identified in each case. They were isolated preparatively, and interspecifically correlated. Subunitα is subdivided into several electrophoretically distinct isoforms which are immunologically closely related (Astacus) or identical (other species). InAstacus andCancer one of these isoforms was shown to dimerize and to act as inter-hexamer bridge. It represents a fourth subunit type termedα′. A fifth, ‘diffuse’ component, which in PAGE migrated at the position of a dimer, was identified in the crossed immunoelectrophoretic patterns as denatured hemocyanin.A common feature of the four hemocyanins is the presence of 4 copies ofβ and 8 copies ofα/γ within the 2×6 particles. Theα:γ ratio is 4:4 in the two Astacidea and 6:2 in the two Brachyura.α′ exists in 2 copies inAstacus andCancer which means that a single dimerα′-α′ is present in a two-hexamer. This leaves 2 monomericα copies inAstacus and 4 inCancer.Every subunit from the four species except ofAstacusα′-α′ was capable to form hexamers in reassembly experiments. If subunit combinations were tested, hetero-hexamers were formed preferentially. Two-hexamers were reconstituted only in the presence of all subunit types and the native subunit stoichiometry was required to obtain twohexamers in considerable yields. Factors limiting 2×6 reassembly are discussed.Authentic 2×6 molecules ofAstacus, Homarus andCancer hemocyanin were immunolabeled with subunit-specific antibody fragments (Fab) or IgG molecules, and the resulting immuno complexes were studied in the electron microscope. A topological model of the quaternary structure of decapod 2×6 hemocyanins is derived, showing the position of each copy of the four subunit types. In this model, the inter-hexamer bridgeα′-α′ is surrounded by twoβ and twoγ subunits forming the central core of the dodecamer. Two additionalβ and two additionalγ subunits form the periphery together with oneα subunit occupying the peripheral short edges of each hexameric half structure. The model is discussed with respect to the current literature.

Comparison of 4 × 6-meric hemocyanins from three different arthropods using computer alignment and correspondence analysis

Abstract We have compared 4 × 6-meric hemocyanin molecules from Androctonus australis and Eurypelma californicum with the similar 4 × 6-meric half-molecules of Limulus polyphemus hemocyanin. This comparison was performed using multivariate statistical analysis applied to computer-aligned electron microscopical images of the molecules: a method that was recently proposed by van Heel & Frank (1980,1981). Our study shows that the molecules are very similar indeed, and that the evidence for a tetrameric arrangement of the hexamers found in 4 × 6-meric Limulus molecules (van Heel & Frank, 1980,1981) also holds for Androctonus and Eurypelma hemocyanin. The model proposed for this molecule by Lamy et al. (1981) is therefore based on correct assumptions. In addition, our study shows that correspondence analysis, as a method of multivariate statistical analysis, is capable of detecting subtle differences between similar structures as well as differences in preparative conditions, effects that were hitherto not accessible to quantitative analysis.

The restriction fragment map of rat-liver mitochondrial DNA: a reconsideration.

Abstract 1. Rat-liver mitochondrial DNA (mtDNA) contains at least 8 cleavage sites for the restriction endonuclease Eco RI, 6 for the restriction endonuclease Hind III, 2 for the restriction endonuclease Bam HI and 11 for the restriction endonuclease Hap II. 2. The physical map of the restriction fragments of Eco RI, Hind III, Bam HI and Hap II is constructed on the basis of: (a) the analysis of partially restricted fragments; (b) analysis of the double digests of total mtDNA; (c) the digestion of isolated restriction fragments with other restriction endonucleases; (d) the identification of fragments of complete single and double digestions and of partially digested fragments containing the base sequences complementary to the 12-S and 16-S RNAs of rat-liver mitochondrial ribosomes. 3. The genes for the ribosomal RNAs are shown to be closely linked. This result differs from data previously reported (Saccone, C., Pepe, G., Cantatore, P., Terpstra, P. and Kroon, A.M. (1976) in The Genetic Function of Mitochondrial DNA, pp. 27–36, Elsevier/North-Holland Biomedical Press, Amsterdam). 4. The origin of replication (D-loop) is localized in the vicinity of the small ribosomal RNA gene and the direction of replication is distant from this gene. 5. The mitochondrial tRNA genes are scattered over the genome as in other animal mtDNAs. The approximate minimal number of tRNA genes is 16–20. 6. We concluded previously that the Eco RI restriction fragments A and D are not adjacent and failed to show the overlap of the 16 S rRNA gene for the Eco RI fragment D and Hind III fragment A. This misinterpretation was due to the fact that the two smallest Eco RI fragments could not be detected with the methods applied and to the lower specific radioactivity of the ribosomal RNAs used in the first series of hybridization experiments.

Calcium-induced fusion of didodecylphosphate vesicles: the lamellar to hexagonal II (HII) phase transition.

SummaryElectron microscopic techniques have been employed to investigate the ability of didodecylphosphate vesicles (diameter approx. 900 Å) to fuse in the presence of Ca2+. As revealed by negative staining, Ca2+ induces extensive fusion and large vesicles with diameters up to 7000 Å are formed. In a processsecondary to fusion, the fused vesicles display a tendency to flatten and are subsequently transformed into extended tubular structures. Freeze-fracture electron microscopy, in conjunction with31P NMR and selected area electron diffraction measurements indicate that the tubes are packed in a hexagonal (HII) array and that the amphiphiles are converted from the lamellar to the hexagonal HII phase.The relationship between membrane fusion and the lamellar-to-hexagonal phase transition is discussed in terms of formation and abundance of transiently stable inverted micellar intermediates at contact regions between two interacting membranes. A model for the conversion of the (vesicular) lamellar into the (tubular) hexagonal HII phase is presented, taking into account the molecular shape of the amphiphile. The relevance of using simple synthetic amphiphiles as models for phospholipid bilayers and complex biomembrane behavior is briefly discussed.

Electron microscopic studies of carboxysomes of Thiobacillus neapolitanus

The outer part of the carboxysomes of Thiobacillus neapolitanus was examined by electron microscopy using negatively stained, cryo-treated, frozen hydrated and freeze dried specimens. From stereo-micrographs of freeze dried and fixated carboxysomes the three dimensional structure of the carboxysomes was elucidated. The carboxysomes always appear as hexagonal bodies, which possess twelve pentameric planes. This indicates that carboxysomes have the form of a pentagonal dodecahedron. Inside the carboxysomes the ribulose-1,5-bisphosphate carboxylase molecules are arranged in rows and concentric rings. Negatively stained and cryo-treated carboxysomes do not differ significantly in size. The mean size of these carboxysomes is 117.3±6.9 nm (n=782)

STRUCTURE OF TETRAHYMENA-PYRIFORMIS MITOCHONDRIAL-DNA .1. STRAIN DIFFERENCES AND OCCURRENCE OF INVERTED REPETITIONS

We have analysed the structure of the mtDNAs of six amicronucleate Tetrahymena pyriformis strains, belonging to at least four phenosets, as defined by Borden et al. (Borden, D., Whitt, G.S. and Nanney, D.L. (1973) J. Protozool. 20, 693--700). 2. The mtDNAs of all strains are linear, but they differ in size, in their fragmentation by endonuclease EcoRI and in overall sequence; less than 20% sequence homology was found by DNA-DNA hybridization in all combinations tested, except for the mtDNAs from strains T and ST which are indistinguishable. 3. In spite of these marked sequence differences the mtDNAs of all strains share two structural peculiarities: ragged (gnawed) duplex ends and a duplication-inversion, which varies in length between 0.3 and 1.2 micrometer, depending on the strain. In four strains the duplication-inversion is terminal, allowing formation of single-stranded DNA circles with a duplex tail; in two strains it is subterminal. 4. The ragged ends and sub-terminal position of the duplication-inversion in some of the Tetrahymena mtDNAs do not fit any of the current models for the replication of linear mtDNAs.

Conservation of the sequence and position of the ribosomal RNA genes in Tetrahymena pyriformis mitochondrial DNA

1. We have done cross-hybridizations between the mitochondrial ribosomal RNAs and DNAs from strains ST and PP of Tetrahymena pyriformis. DNA . ribosomal RNA hybrid formation can be completely prevented by an excess of the heterologous ribosomal RNA and the heterologous hybrids melt 6 degrees C below the homologous hybrids. This shows that the ribosomal RNA cistrons can account for the 5% cross-hybridization previously observed between the mtDNAs of strains PP and ST (Goldbach et al. (1977) Biochim. Biophys. Acta 477, 37--50). 2. By electron microscopy of DNA . ribosomal RNA hybrids we have determined the position of the ribosomal RNA cistrons on the mtDNA of strain GL, a mtDNA which we have shown to contain a sub-terminal 1 micron duplication-inversion and a terminal palindrome at one end which varies in length from 0 to 5 micron and which includes the 1 micron duplication-inversion (Arnberg et al. (1977) Biochim. Biophys. Acta 477, 51--69). The 21 S ribosomal RNA cistron overlaps the 1 micron duplication-inversion and as a result two or three cistrons are present, depending on the size of the terminal palindrome. Only one 14 S ribosomal RNA cistron is found, located about 10 000 base pairs away from the nearest 21 S cistron is found, located about 10 000 base pairs away from the nearest 21 S cistron and with the same polarity as this cistron. 3. We conclude from these results and those in the preceding paper that the sequence of the ribosomal RNAs and the position of the ribosomal RNA genes in the mtDNA is strongly conserved in Tetrahymena. Possible reasons for the duplication of 21-S ribosomal RNA genes and the terminal heterogeneity of Tetrahymena mtDNA are discussed.

Mitochondrial DNA of Tetrahymena pyriformis strain ST contains a long terminal duplication-inversion

1. We have studied denatured Tetrahymena mtDNA by electron microscopy using the formamide technique. 2. After denaturation all DNA is single stranded, but within a few minutes single-stranded circles with a duplex tail are formed. 3. The duplex tail is 1.3 mum long, i.e. 8 percent of the length of native mtDNA, and it often contains a small single-stranded eye. 4. Digestion of the duplex DNA with exonuclease III of Escherichia coli abolishes its ability to form circles and duplex tails after denaturation. 5. Renaturation of denatured mtDNA leads to the formation of duplex circles with single-stranded section and/or duplex tails. In addition, a minority of duplex circles without apparent tails is formed, but these circles contain a small ambiguous section. 6. We conclude that this mtDNA contains a long terminal duplication-inversion, that could be involved in the replication of this linear mtDNA.