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  • 1
    ISSN: 1432-2048
    Keywords: Allium ; Cell cycle ; DNA synthesis inhibitors and cell cycle ; Microtubule ; Preprophase band ; Root meristem
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, microspectrophotometry of nuclear DNA, and autoradiography of [3H]thymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6-10-fold to less than 0.5% when roots were kept in 20 μg·ml-1 aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 μg·ml-1 5-aminouracil and 40 μg·ml-1 hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1615-6102
    Keywords: Basal body ; Cytoskeleton ; Microtubule ; Immunoflu orescence ; Monoclonal antibodies ; Pteridium ; Spermatozoid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Detergent extracted spermatozoids of the fernPteridium aquilinum were used as mixed antigen preparations for raising monoclonal antibodies in order to obtain reagents for detecting as yet uncharacterized components of the plant cytoskeleton. Selected antibodies were studied by immunofluorescence microscopy of developing spermatids and mature spermatozoids. Some reacted directly with fixed cells, others required permeabilization treatments with cold methanol or Triton X-100. AntibodiesPas2D9 andPas6D7 bind to glycoprotein antigenic determinants that are exposed on the surface of the plasma membrane. Several antibodies interact with cytoskeletal components.Pas1D3,Pas5D8 andPas5F4 bind to the cytoskeleton of permeabilized cells including the flagella. Three react specifically with the flagellar band or associated components:Pas2G6 reacts with the whole flagellar band but shows a prominent binding to basal bodies,Pas5E2 binds exclusively to basal bodies, andPas5E7 detects mitochondria associated with the flagellar band. Cross-reactions to wheat root tip cells at different stages of the cell cycle are described inMarc andGunning (1988).
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  • 3
    ISSN: 1615-6102
    Keywords: Immunoglobulin G ; Immunoglobulin Y ; Plant tubulin ; Tubulin antibodies ; Tubulin isoforms ; Zea mays
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Antibodies specific to five different maize isotubulins were made. From predicted amino acid sequences established from previously sequenced maize tubulin genes, peptide antigens were synthesized matching the carboxyl-terminal 11–13 amino acids of each of three maize α-tubulins and two maize β-tubulins. Antibodies were generated by injecting conjugated antigens into hens, collecting their eggs, and extracting immunoglobulin Y from the egg yolk. Specificity of each antibody was tested by immunoblotting of fusion proteins containing the antigenic sequence of the specific α- and β-tubulin isoforms. For all five isotubulins, antibodies were affinity-purified with fusion proteins corresponding to their respective antigens, to remove nonspecific binding found in the antibody preparations. Further preparation of anti-α-tubulins was required to eliminate cross-reactivity of antibodies with members of other α-tubulin subfamilies. For this, affinity-purified antibodies against a specific α-tubulin were preadsorbed with peptides representing cross-reactive α-tubulin antigens. Results indicated that virtually all cross-reactivity between members of different α-tubulin subfamilies could be eliminated, resulting in labeling of only the fusion protein containing the specific antigen. All five isotubulin antibodies generated showed labeling of discrete spots on two-dimensional immunoblots of maize proteins, demonstrating the specificity of the antibodies in complex tubulin mixtures. These antibodies should prove valuable for analyzing the developmental distribution, and possible functional significance, of several maize isotubulins.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 204 (1998), S. 235-244 
    ISSN: 1615-6102
    Keywords: Immunofluorescence microscopy ; Plant microtubules ; Tubulin antibodies ; Tubulin isoforms ; Zea mays
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Antibodies specific to two of the maize β-tubulin isoforms and to the three subfamilies of maize α-tubulins were used in immunofluorescence microscopy to determine where and into which microtubule (MT) arrays these tubulin isoforms are incorporated in maize plants. All the tubulins examined appear to be incorporated into MTs in at least some cell types, with the possible exception of subfamily II α-tubulins, which have been found only in the form of diffuse, nonfibrillar staining. Whereas the α-tubulins of subfamily I appear to be used constitutively, others are used much more selectively in the plant, with β2-tubulin found in microtubules only during sexual reproduction. If a particular tubulin is used in the MTs of a given cell type, it appears to be incorporated into all the MT arrays found in the cell.
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  • 5
    Publication Date: 1988-01-01
    Print ISSN: 0032-0935
    Electronic ISSN: 1432-2048
    Topics: Biology
    Published by Springer
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