Identification and characterization of axopodial tubulins from Echinosphaerium nucleofilum

Isolated microtubule protein from axopodia of the heliozoan Echinosphaerium nucleofilum, consisting of two major bands on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), has been compared to axonemal and cytoplasmic tubulins from both animal and non-animal sources. The upper E. nucleofilum protein band migrated faster than the alpha-tubulins of bovine brain and sea anemone sperm tails but with approximately the same electrophoretic mobility as the axonemal alpha-tubulins of Tetrahymena pyriformis and the alga Chlorogonium elongatum and cytoplasmic alpha-tubulin from the slime mold Physarum polycephalum. The lower E. nucleofilum protein band, however, had a higher electrophoretic mobility than all the beta-tubulins which we have so far examined. It was, nevertheless, a true beta-tubulin as shown by its migration on two-dimensional gel electrophoresis and the general resemblance of its one- and two-dimensional peptide maps to those of other beta-tubulins. The Staphylococcus aureus protease cleavage pattern of the upper axopodial protein band was similar to those of other non-animal alpha-tubulins but quite different from those of the animal alpha-tubulins. In contrast, the two-dimensional tryptic peptide map of axopodial alpha-tubulin was distinct from all of them. For example, a characteristic constellation of peptides common to the peptide maps of the other alpha-tubulins was absent from that of E. nucleofilum. In contrast to Physarum and metazoan tubulins but similar to Tetrahymena tubulin, the axopodial alpha-tubulin had a more basic isoelectric point than the beta-subunit as shown by two dimensional gel electrophoresis. Some of the unusual characteristics of E. nucleofilum axopodial tubulin may not only reflect phylogenetic variation, but also the different functional requirements of axopodial microtubules.     

Motility in Echinosphaerium nucleofilum. II. Cytoplasmic contractility and its molecular basis

Echinosphaerium nucleofilum exhibits at least three kinds of movement: locomotion by the bending and shortening of its many axopodia, feeding by means of food-cup pseudopodia formed from its cortical cytoplasm, and saltatory motion of cytoplasmic particles, especially in the cortex and axopodia. Since previously presented evidence indicated that the microtubular axoneme is not essential for particle motion, the cytoplasm was investigated for the possible existence of contractile behavior and for the possible presence of linear elements other than microtubules. Cytoplasm can be isolated in physiological media in which rigor, relaxation, and contraction can be induced, as in muscle, by manipulating the concentrations of calcium ions and magnesium-adenosine triphosphate. Contraction is initiated by calcium ions at concentrations above 2.4 times 10-minus 7 M. The rigor-to-relaxation transition occurs at subthreshold calcium concentrations on the addition of 10-minus 3 M ATP. Negatively stained preparations of isolated cytoplasm show two types of filaments: thin filaments identified as cytoplasmic actin by virtue of their binding heavy meromyosin from striated muscle in characteristic arrowhead arrays, and thicker filaments which do not strictly resemble myosin aggregates from muscle or amoeba but could conceivably by myosin aggregated in an unfamiliar form.     

Structure and Elemental Composition of the Cyst Wall of Echinosphaerium nucleofilum Barrett (Heliozoea,Actinophryida)1

The structure of the cyst wall of the heliozoon Echinosphaerium nucleofilum has been investigated using light microscopy, scanning and transmission electron microscopy, and X-ray microanalysis. The cyst wall is a composite structure of seven or eight layers. These are: an enveloping gelatinous layer; a layer of siliceous spheroidal bodies; an electron-dense supporting membrane; a broad electron-lucent zone; an electron-dense layer; a layer of helicoidally packed material; and one or two layers with a granular appearance lying next to the plasma membrane of the encysted organism. The structure of the cyst wall closely resembles that of Actinophrys sol, confirming the close relationship of these actinophryid heliozoa while emphasizing their distinctiveness from other amoeboid protista.     

Motility in Echinosphaerium nucleofilum. I. An analysis of particle motions in the axopodia and a direct test of the involvement of the axoneme

The motion of particles in the axopodia of Echinosphaerium nucleofilum is saltatory. In the present study, photokymograph records of 123 motions from six axopodia have been analyzed. Particles followed rectilinear paths of from 1 to 15 mum while in continuous motion at an average velocity of 0.66 plus or minus 0.32 mum/s. The velocity of the particles was variable in 36% of the cases measured. Some motions were punctuated by pauses either before continuing in the same direction or reversing. Frequently, several particles were moving at the same velocity, but neighboring particles showed no motion or moved in the opposite direction. Two particles occasionally contacted one another and travelled as a unit for varying lengths of time but subsequently moved independently. These motions reflect the underlying mechanism of motive force production. Furthermore, a glass microneedle can be substituted for the microtubular axoneme in the axopodia. In these artificial axopodia, bidirectional particle motions occurred which were similar to those in normal axopodia. Colchicine, at the threshold dose for axonemal dissolution, had no affect on these particel motions. It is concluded that the microtubular axoneme is not responsible for particle motions and also that individual microtubules are unlikely candidates for motive force production in this system.     

Diameter Oscillation of Axonemes in Sea-Urchin Sperm Flagella

The 9 + 2 configuration of axonemes is one of the most conserved structures of eukaryotic organelles. Evidence so far has confirmed that bending of cilia and flagella is the result of active sliding of microtubules induced by dynein arms. If the conformational change of dynein motors, which would be a key step of force generation, is occurring in a three-dimensional manner, we can easily expect that the microtubule sliding should contain some transverse component, i.e., a motion in a direction at a right angle to the longitudinal axis of axonemes. Using a modified technique of atomic force microscopy, we found such transverse motion is actually occurring in an oscillatory manner when the axonemes of sea-urchin sperm flagella were adhered onto glass substrates. The motion was adenosine triphosphate-dependent and the observed frequency of oscillation was similar to that of oscillatory sliding of microtubules that had been shown to reflect the physiological activity of dynein arms (S. Kamimura and R. Kamiya. 1989. Nature. 340:476–478; 1992. J. Cell Biol. 116:1443–1454). Maximal amplitude of the diameter oscillation was around 10 nm, which was within a range of morphological change observed with electron microscopy (F. D. Warner. 1978. J. Cell Biol. 77:R19–R26; N. C. Zanetti, D. R. Mitchell, and F. D. Warner. 1979. J. Cell Biol. 80:573–588).     

Flagellar Axonemes with 10 Microtubular Doublets in Spermatozoa from Gall-midges (Diptera,Cecidomyiidae)

Abstract The spermatozoa of some gall-midges (Cecidomyiidae, Lestremiinae), belonging to the tribe Micromyini, were seen to have an axoneme that consists of 10, instead of nine, microtubular doublets surrounding a central cylinder. In some related species within the same tribe the axoneme was found to contain a similar cylinder but to have nine doublets, as in typical flagella, or to have nine doublets and no central structure. These three types of axonemes can be given the shorthand designations “10+cyl”,“9+cyl”, and “9+0”. The tribe Lestremiini is characterized by a giant axoneme having 150 doublets in two rows reversely oriented. Other characteristics of examined spermatozoa are the electron density of the B-tubules of the axoneme, a feature shared by all members of the subfamily Lestremiinae, and the presence of a prominent cytoplasmic droplet containing numerous, regularly spaced microtubules, which is shared by all Micromyidi. These axonemal models are discussed from a phylogenetic point of view.     

Analyses of 22S Dynein Binding to Tetrahymena Axonemes Lacking Outer Dynein Arms

ABSTRACT. Tetrahymena thermophila mutants homozygous for the oad mutation become nonmotile when grown at the restrictive temperature of 39° C. Axonemes isolated from nonmotile oad mutants ( oad 39° C axonemes) lack approximately 90% of their outer dynein arms and are deficient in 22S dynein. Here we report that oad 39° C axonemes contain 40% of the 22S dynein heavy chains that wild-type axonemes contain and that oad axonemes do not undergo ATP-induced microtubule sliding in vitro. Wild-type 22S dynein will bind to the outer arm position in oad axonemes and restore ATP-induced microtubule sliding in those axonemes. Unlike wild-type 22S dynein, oad 22S dynein does not bind to the outer arm position in oad axonemes. These data indicate that the oad mutation affects some component of the outer arm dynein itself rather than the outer arm dynein binding site. These data also indicate that oad axonemes can be used to assay outer dynein arm function.     

水稻OsGSTL1启动子的分离和表达分析

从水稻基因组文库中筛选得到一个水稻GST基因,命名为OsGSTL1.半定量RT-PCR分析表明OsGSTL1基因的表达不受绿磺隆、乙烯利、脱落酸、水杨酸和茉莉酸甲酯的诱导,因此该基因可能与植物抗逆性无关.为了研究OsGSTL1启动子在植物体内的表达特性,将OsGSTL1起始位点5＇端上游不同长度的调控序列与报告基因GUS融合,并在洋葱表皮瞬间表达和拟南芥中稳定表达.研究表明：在洋葱表皮细胞中,160bp及更长的上游调控序列均能启动GUS基因的表达;而在转基因拟南芥中,含有2155 bp的上游序列的PGZ2.1：：GUS具有时空表达的特性,在转基因的早期幼苗中GUS基因在子叶中特异性表达,但在根中没有表达;而在幼苗生长的后期,根、茎、叶中都有少量的表达.但包含1 224 bp的上游序列的PGZ1.2：：GUS却表现为组成型表达的特性.由此推测,OsGSTL1启动子启动的基因表达可能与幼苗的营养代谢相关;而OsGSTL1启动子的时空表达相关元件可能位于OsGSTL1翻译起始位点5＇端上游-2155 bp至-1224 bp范围内.     

Isolation and Properties of Macronuclei from Tetrahymena thermophila1

A simple and efficient method is described for the isolation of macronuclei from Tetrahymena thermophila (7B). The steps involved are deciliation and removal of the mucocysts’ contents by dibucaine treatment, digitonin mediated lysis, differential centrifugations, and finally isopyenic sucrose density gradient centrifugation. Judging from the distribution of marker enzymes and electron microscopy, the macronuclei obtained were free of cytoplasmic and paniculate contamination and were highly active in endogenous RNA-synthesis (1.5 pmol UTP incorporation/ng DNA min at 30°C). The ratio of protein: RNA: DNA was 2.0:0.33:1.0 (weight) and each macronucleus contained an average of 17 pg DNA. The average yield of isolation was 50%.     

Isolation of Legionella longbeachae serogroup 1 from potting mixes

Following a statewide outbreak of legionellosis due to Legionella longbeachae serogroup 1 in South Australia in 1988 and 1989, studies were performed to find a source of the organism. A number of water and soil samples with and without acid decontamination were examined for L. longbeachae by using a selective medium containing vancomycin, aztreonam, and pimafucin. There were no isolations of L. longbeachae from water samples. Organisms resembling L. longbeachae were isolated from a number of samples of potting mixes and from soil surrounding plants in pots collected from the homes of four patients. The organisms were found to persist for 7 months in two potting mixes stored at room temperature. Legionellae were isolated with difficulty from potting mixes which were allowed to dry out. Identification of isolates as L. longbeachae serogroup 1 was confirmed by quantitative DNA hybridization and serological tests. Restriction-fragment-length-polymorphism studies showed minor differences between patient and environmental isolates but differentiated these readily from L. longbeachae serogroup 2 and other antigenically related legionellae. The isolation of L. longbeachae from some potting mixes and the prolonged survival of the organisms in this medium suggest that soil rather than water is the natural habitat of this species and may be the source of human infections.     

Isolation of the cytochrome-bc1 complex from rat-liver mitochondria

The cytochrome bc1 complex has been isolated from rat-liver mitochondria by two different procedures. The enzyme isolated by either procedure exhibits a specific cytochrome b and cytochrome c1 heme content of approximately 8 and 4 nmol/mg protein respectively. Both preparations contain only seven polypeptides on sodium dodecylsulfate gel electrophoresis, with the following apparent molecular weights: I, 50000; II, 46000; III, 33000; IV, 25000; V, 12500; VI, 10000; VII, 5600. The polypeptide composition is identical to that of the beef-heart enzyme isolated by cholate/ammonium sulfate fractionation. Furthermore, with the exception of subunit II (core protein 2), the apparent molecular weights of the subunits are identical in the rat-liver and beef-heart enzymes.     

A model of contractile tubules showing how they contract in the heliozoan Echinosphaerium

In the large heliozoan Echinosphaerium, contractile tubules (formerly called X-bodies) are located between the axopodial membrane and the axonemal microtubules. When axopodial contraction occurs, the tubules have been thought to be transformed from a tubular to a granular form, as seen in ultra-thin sections. Our detailed morphological observations of the contractile tubules, however, have revealed that this so-called granulation of the contractile tubules is mediated by self-twisting and supercoiling during contraction. We also examined the localization of calcium during axopodial contraction using a potassium pyroantimonate assay. Ca-Sb deposits were detected on contractile tubules only during the twisting and coiling of this organelle. Our results indicate that axopodial contraction is enforced by the twisting and coiling of contractile tubules, which action probably is mediated by Ca2+ ions.     

Isolation of Legionella longbeachae serogroup 1 from potting mixes.

Following a statewide outbreak of legionellosis due to Legionella longbeachae serogroup 1 in South Australia in 1988 and 1989, studies were performed to find a source of the organism. A number of water and soil samples with and without acid decontamination were examined for L. longbeachae by using a selective medium containing vancomycin, aztreonam, and pimafucin. There were no isolations of L. longbeachae from water samples. Organisms resembling L. longbeachae were isolated from a number of samples of potting mixes and from soil surrounding plants in pots collected from the homes of four patients. The organisms were found to persist for 7 months in two potting mixes stored at room temperature. Legionellae were isolated with difficulty from potting mixes which were allowed to dry out. Identification of isolates as L. longbeachae serogroup 1 was confirmed by quantitative DNA hybridization and serological tests. Restriction-fragment-length-polymorphism studies showed minor differences between patient and environmental isolates but differentiated these readily from L. longbeachae serogroup 2 and other antigenically related legionellae. The isolation of L. longbeachae from some potting mixes and the prolonged survival of the organisms in this medium suggest that soil rather than water is the natural habitat of this species and may be the source of human infections.