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    BASAL BODY REORIENTATION MEDIATED BY A CA-2+-MODULATED CONTRACTILE PROTEIN
    MCFADDEN, GI ; SCHULZE, D ; SUREK, B ; SALISBURY, JL ; MELKONIAN, M (ROCKEFELLER UNIV PRESS, 1987-08-01)
    A rapid, Ca2+-dependent change in the angle between basal bodies (up to 180 degrees) is associated with light-induced reversal of swimming direction (the "photophobic" response) in a number of flagellated green algae. In isolated, detergent-extracted, reactivated flagellar apparatus complexes of Spermatozopsis similis, axonemal beat form conversion to the symmetrical/undulating flagellar pattern and basal body reorientation (from the antiparallel to the parallel configuration) are simultaneously induced at greater than or equal to 10(-7) M Ca2+. Basal body reorientation, however, is independent of flagellar beating since it is induced at greater than or equal to 10(-7) M Ca2+ when flagellar beating is inhibited (i.e., in the presence of 1 microM orthovanadate in reactivation solutions; in the absence of ATP or dithiothreitol in isolation and reactivation solutions), or when axonemes are mechanically removed from flagellar apparatuses. Although frequent axonemal beat form reversals were induced by varying the Ca2+ concentration, antiparallel basal body configuration could not be restored in isolated flagellar apparatuses. Observations of the photophobic response in vivo indicate that even though the flagella resume the asymmetric, breaststroke beat form 1-2 s after photostimulation, antiparallel basal body configuration is not restored until a few minutes later. Using an antibody generated against the 20-kD Ca2+-modulated contractile protein of striated flagellar roots of Tetraselmis striata (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, 1984, J. Cell Biol., 99:962-970), we have found the distal connecting fiber of Spermatozopsis similis to be immunoreactive by indirect immunofluorescence and immunogold electron microscopy. Electrophoretic and immunoblot analysis indicates that the antigen of S. similis flagellar apparatuses consists, like the Tetraselmis protein, of two acidic isoforms of 20 kD. We conclude that the distal basal body connecting fiber is a contractile organelle and reorients basal bodies during the photophobic response in certain flagellated green algae.
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    Ultraviolet microbeam irradiations of mitotic diatoms: investigation of spindle elongation.
    Leslie, RJ ; Pickett-Heaps, JD (Rockefeller University Press, 1983-02)
    Our simple instrumentation for generating a UV-microbeam is described UV microbeam irradiations of the central spindle in the pennate diatom Hantzschia amphioxys have been examined through correlated birefringence light microscopy and TEM. A precise correlation between the region of reduced birefringence and the UV-induced lesion in the microtubules (MTs) of the central spindle is demonstrated. The UV beam appears to dissociate MTs, as MT fragments were rarely encountered. The forces associated with metaphase and anaphase spindles have been studied via localized UV-microbeam irradiation of the central spindle. These spindles were found to be subjected to compressional forces, presumably exerted by stretched or contracting chromosomes. Comparisons are made with the results of other writers. These compressional forces caused the poles of a severed anaphase spindle to move toward each other and the center of the cell. As these poles moved centrally, the larger of the two postirradiational central spindle remnants elongated with a concomitant decrease in the length of the overlap. Metaphase spindles, in contrast, did not elongate nor lose their overlap region. Our interpretation is that the force for anaphase spindle elongation in Hantzschia is generated between half-spindles in the region of MT overlap.
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    UV microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production.
    Spurck, TP ; Stonington, OG ; Snyder, JA ; Pickett-Heaps, JD ; Bajer, A ; Mole-Bajer, J (Rockefeller University Press, 1990-10)
    Metaphase and anaphase spindles in cultured newt and PtK1 cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation. After this enlargement of the ARB, metaphase spindles recovered rapidly as the detached pole moved back towards the chromosomes, reestablishing spindle fibers as the ARB closed; this happened when the ARB cut a few fibers or across the entire half spindle. We never detected elongation of the cut kinetochore fibers. Rather, astral fibers growing from the pole appeared to bridge and then close the ARB, just before the movement of the pole toward the chromosomes. When a second irradiation was directed into the closing ARB, the polewards movement again stopped before it restarted. In all metaphase cells, once the pole had reestablished connection with the chromosomes, the unirradiated half spindle then also shortened to create a smaller symmetrical spindle capable of normal anaphase later. Anaphase cells did not recover this way; the severed pole remained detached but the chromosomes continued a modified form of movement, clumping into a telophase-like group. The results are discussed in terms of controls operating on spindle microtubule stability and mechanisms of mitotic force generation.
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    Cell division in two large pennate diatoms Hantzschia and Nitzschia III. A new proposal for kinetochore function during prometaphase.
    Tippit, DH ; Pickett-Heaps, JD ; Leslie, R (Rockefeller University Press, 1980-08)
    Prometaphase in two large species of diatoms is examined, using the following techniques: (a) time-lapse cinematography of chromosome movements in vivo; (b) electron microscopy of corresponding stages: (c) reconstruction of the microtubules (MTs) in the kinetochore fiber of chromosomes attached to the spindle. In vivo, the chromosomes independently commence oscillations back and forth to one pole. The kinetochore is usually at the leading edge of such chromosome movements; a variable time later both kinetochores undergo such oscillations but toward opposite poles and soon stretch poleward to establish stable bipolar attachment. Electron microscopy of early prometaphase shows that the kinetochores usually laterally associate with MTs that have one end attached to the spindle pole. At late prometaphase, most chromosomes are fully attached to the spindle, but the kinetochores on unattached chromosomes are bare of MTs. Reconstruction of the kinetochore fiber demonstrates that most of its MTs (96%) extend past the kinetochore and are thus apparently not nucleated there. At least one MT terminates at each kinetochore analyzed. Our interpretation is that the conventional view of kinetochore function cannot apply to diatoms. The kinetochore fiber in diatoms appears to be primarily composed of MTs from the poles, in contrast to the conventional view that many MTs of the kinetochore fiber are nucleated by the kinetochore. Similarly, chromosomes appear to initially orient their kinetochores to opposite poles by moving along MTs attached to the poles, instead of orientation effected by kinetochore MTs laterally associating with other MTs in the spindle. The function of the kinetochore in diatoms and other cell types is discussed.
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    Organization of spindle microtubules in Ochromonas danica.
    Tippit, DH ; Pillus, L ; Pickett-Heaps, J (Rockefeller University Press, 1980-12)
    The entire framework of microtubules (MTs) in the mitotic apparatus of Ochromonas danica is reconstructed (except at the spindle poles) from transverse serial sections. Eleven spindles were sectioned and used for numerical data, but only four were reconstructed: a metaphase, an early anaphase, a late anaphase, and telophase. Four major classes of MTs are observed: (a) free MTs (MTs not attached to either pole); (b) interdigitated MTs (MTs attached to one pole which laterally associate with MTs from the opposite pole); (c) polar MTs (MTs attached to one pole); (d) kinetochore MTs (kMTs). Pole-to-pole MTs are rare and may be caused by tracking errors. During anaphase, the kMTs, free MTs, and polar MTs shorten until most disappear, while interdigitated MTs lengthen. In the four reconstructed spindles, the number of MTs decreases between early anaphase and telophase from 881 to 285, while their average length increases from 1.66 to 4.98 micron. The total length of all the MTs in the spindle (placed end to end) remains at 1.42 +/- 0.04 mm between these stages. At late anaphase and telophase the spindle is comprised mainly of groups of interdigitated MTs. Such MTs from opposite poles form a region of overlap in the middle of the spindle. During spindle elongation (separation of the poles), the length of the overlap region does not decrease. These results are compatible with theories that suggest that MTs directly provide the force that elongates the spindle, either by MT polymerization alone or by MT sliding with concomitant MT polymerization.
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    Chromosome motion and the spindle matrix.
    Pickett-Heaps, J ; Spurck, T ; Tippit, D (Rockefeller University Press, 1984-07)
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    On the mechanism of anaphase A: evidence that ATP is needed for microtubule disassembly and not generation of polewards force.
    Spurck, TP ; Pickett-Heaps, JD (Rockefeller University Press, 1987-10)
    As anaphase began, mitotic PtK1 and newt lung epithelial cells were permeabilized with digitonin in permeabilization medium (PM). Permeabilization stopped cytoplasmic activity, chromosome movement, and cytokinesis within about 3 min, presumably due to the loss of endogenous ATP. ATP, GTP, or ATP-gamma-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened. AMPPNP could not restart anaphase A; ATP was ineffective if the spindle was stabilized in PM + DMSO. Cells permeabilized in PM + taxol varied in their response to ATP depending on the stage of anaphase reached: one mid-anaphase cell showed initial movement of chromosomes back to the metaphase plate upon permeabilization but later, anaphase A resumed when ATP was added. Anaphase A was also reactivated by cold PM (approximately 16 degrees C) or PM containing calcium (1-10 mM). Staining of fixed cells with antitubulin showed that microtubules (MTs) were relatively stable after permeabilization and MT assembly was usually promoted in asters. Astral and kinetochore MTs were sensitive to MT disassembly conditions, and shortening of kinetochore MTs always accompanied reactivation of anaphase A. Interphase and interzonal spindle MTs were relatively stable to cold and calcium until extraction of cells was promoted by longer periods in the PM, or by higher concentrations of detergent. Since we cannot envisage how both cold treatment or relatively high calcium levels can reactivate spindle motility in quiescent, permeabilized, and presumably energy-depleted cells, we conclude that anaphase A is powered by energy stored in the spindle. The nucleotide triphosphates effective in reactivating anaphase A could be necessary for the kinetochore MT disassembly without which anaphase movement cannot proceed.
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    The analysis of quantitative variation in natural populations with isofemale strains
    HOFFMANN, AA ; PARSONS, PA (BioMed Central, 1988-03-01)
    Isofemale strains are having an increasing role in the analysis of variability of ecological and behavioural traits in natural populations. This paper therefore considers the association between heritability and phenotypic variation within and between isofemale strains. Heritability from an isofemale strain analysis approximates narrow heritability over a wide range of dominance values, particularly when genes contributing to variation in a trait are at intermediate frequencies. Meaningful heritability estimates require that isofemale strains are maintained at a population size greater than 50 and tested within 5 generations after establishment. Values of heritabilities for morphological traits in Drosophila melanogaster were similar to those estimated from a conventional sib analysis. Published data on isofemale strains can therefore be put into a theoretical framework. The contribution of isofemale strain analyses to the debate about the number of loci affection variation in quantitative traits is briefly discussed.
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    Population-genetics of the metabolically related Adh, Gpdh and Tpi polymorphisms in Drosophila melanogaster: II. Temporal and spatial variation in and orchard population
    NIELSEN, KM ; HOFFMANN, AA ; MCKECHNIE, SW (BioMed Central, 1985-01-01)
    Seasonal and spatial variation in gene frequencies at 3 diallelic loci : alcohol dehydrogenase (Adh), glycerophosphate dehydrogenase (Gpdh) and triosephosphate isomerase (Tpi), have been studied in an orchard population of D. melanogaster. Gene frequency at the Tpi locus varied seasonally and was associated positively with total monthly rainfall measured both immediately prior to and concurrent with the month of collection. Temporal herteogeneity, not associated with the environmental parameters, was present at the Adh locus. Gpdh-F frequency was negatively associated with mean monthly maximum temperature measured prior to the time of collection. Within the orchard site, spatial heterogeneity in gene frequency at the Tpi locus was observed within collections. a deficiency of Gpdh heterozygotes was observed in individual trap samples and among collections with traps pooled. Overall, this variation is interpreted as being due to sampling from a population of partially isolated subgroups, founded by few individuals, and dependent upon transient pockets of fruit resources.
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    Bidirectional selection for olfactory response to acetaldehyde and ethanol in Drosophila melanogaster
    HOFFMANN, AA (BioMed Central, 1983-01-01)
    Drosophila melanogaster adults were successfully selected for increased and decreased olfactory response to different concentrations of acetaldehyde and ethanol, using a choice type olfactometer. The response to selection was not concentration specific. Lines were also tested with n-propanol, n-butanol, acetic acid, propionic acid, benzaldehyde, methyl acetate, ethyl acetate and 3-hydroxybutan-2-one. The behavior of the lines was similar for most of these chemicals, indicating an odorant specific response to selection. However, ethanol selected lines differed in their response to n-propanol and possibly n-butanol, suggesting a role for the alcohol group in determining the specificity of genetic variation in olfactory response. Utilization of acetic acid, ethanol and acetaldehyde as resources by the selected lines was tested using adult longevity. The only significant result was that lines selected for increased attraction to ethanol ulitised 9 p. 100 and 12 p. 100 ethanol more than lines selected for decreased attraction. Gene frequencies were monitored at 3 enzyme loci – alcohol dehydrogenase, triose phosphate isomerase, α-glycerophosphate dehydrogenase – associated with the metabolism of ethanol and acetaldehyde. There were no changes associated with the metabolism of ethanol and acetaldehyde. There were no changes associated with the response to selection, and the ADH-F allele increased in both selected and unselected lines.