American Journal of Medical Genetics 83:334–337 (1999) Letter to the Editor First Transmission Electron Micrograph of Continuous Mitotic Spindle Fibers Between Polar Area and Chromosome Ends To the Editor: During the study of the ultrastructure of the fragile X chromosome reported in these proceedings [Wen et al., 1999], one striking preparation demonstrated an apparent mitotic spindle apparatus not visible in the Q-banded metaphase (Fig. 1) composed of microtubules with diameters of 200 to 260 Å (Figs. 2 and 3). Two microtubules per spindle fiber were observed (Fig. 3). An apparent polar area or centriole was also observed. These apparent microtubules were thought to be distinct from chromatin fibers based on their characteristic size and configuration and also because none of the apparent spindle apparatus was visible in the DNAspecific Q-banded preparation (Fig. 1). This appears to be the first such transmission electron microscope (TEM) observation of the mitotic spindle apparatus with continuous microtubules forming loops and connecting the ends of the short arms of both X and number 4 chromosomes as illustrated in Fig. 4. The apparent mitotic spindle apparatus in Figs. 2 to 4, to our knowledge, is the first TEM observation of a monoplanar longitudinal section of metaphase chromosomes connected from their short arms to microtubules. Furthermore, it appears more likely that what we have observed in Fig. 3 is part of the mitotic spindle apparatus rather than chromatin fibers stretching from the chromosome [Hliscs et al., 1997] because of the following: 1) chromatin fibers appear to be flexible and have a ‘‘zig-zag’’ configuration, whereas spindle microtubule fibers appear more rigid and straight [Horowitz et al., 1994; Brinkley, 1997]; 2) chromatin fiber diameter has been established at 300 Å [Ostashevsky and Lange, 1994; Horowitz et al., 1994; Bartolome et al., 1994; Santisteban, 1994; Martin et al., Contract grant sponsor: New York State Office of Mental Retardation and Developmental Disabilities; Contract grant sponsor: the Maternal and Child Health Program (Title V, Social Security Act), Health Resources and Services Administration, Department of Health and Human Services; Contract grant number: MCJ360587. *Correspondence to: Dr. Guang Wen, Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314. E-mail: email@example.com Received 22 August 1997; Accepted 9 November 1998 © 1999 Wiley-Liss, Inc. 1995], whereas microtubules have been shown to be 200 to 260 Å [Brinkley, 1997]; and 3) the Q-banded metaphase did not exhibit this spindle-like configuration coming from the X and number 4 chromosomes. ACKNOWLEDGMENTS We thank Dr. James Ray of Quest Laboratories for constructive criticism during the preparation of this manuscript. This work was supported in part by the New York State Office of Mental Retardation and Developmental Disabilities and in part by grant MCJ360587 from the Maternal and Child Health Program (Title V, Social Security Act), Health Resources and Services Administration, Department of Health and Human Services. REFERENCES Bartolome S, Bermudez A, Daban JR. 1994. Internal structure of the 30 nm chromatin fiber. J Cell Sci 107:2983–2992. Brinkley W. 1997. Microtubules: A brief historical perspective. J Struct Biol 118:84–86. Hliscs R, Muhlig P, Claussen U. 1997. The spreading of metaphases is a slow process which leads to a stretching of chromosomes. Cytogenet Cell Genet 76:167–171. Horowitz RA, Agar DA, Sedat JW, Woodcock CL. 1994. The threedimensional architecture of chromatin in situ: electron tomography reveals fibers composed of a continuously variable zig-zag nuleosomal ribbon. J Cell Biol 125:1–10. Martin LD, Vesenka JP, Henderson E, Dobbs DL. 1995. Visualization of nucleosomal substructure in native chromatin by atomic force microscopy. Biochem 34:4610–4616. Ostashevsky JY, Lange CS. 1994. The 30 nm chromatin fiber as a flexible polymer. J Biolmol Struct Dyn 11:813–820. Santisteban MS. 1994. Structure of chromatin I: Levels of DNA organization in the nucleus: nucleosome and chromatin fibers. Pathol Biol 42: 868–883. Wen GY, Jenkins EC, Goldberg EM, Genovese M, Brown WT, Wisniewski HM. 1999. Ultrastructure of the fragile X chromosome: new observations on the fragile site. Am J Med Genet 83:331–333. Guang Y. Wen* Edmund C. Jenkins Erick M. Goldberg Marilyn Genovese W. Ted Brown Henryk M. Wisniewski Institute for Basic Research in Developmental Disabilities Staten Island, New York Letter to the Editor 335 Fig. 1. Fluorescent light micrograph of a control pre-identified X chromosome (arrow) after Q-banding (DNA-specific) showing no indication of spindle fibers (×3,048). Fig. 2. Electron micrograph of the same X chromosome as in Fig. 1, showing an apparent part of the mitotic spindle apparatus connecting both X and chromosome 4 at their distal ends to an apparent polar region (centriole) (×8,285). 336 Wen et al. Fig. 3. Higher magnification electron micrograph of the same mitotic spindle apparatus shown in Figure 2, revealing continuous microtubule fibers between X and chromosome 4 short arm telomeres and the centriole (×18,657). Two parallel microtubules (see insert, ×123,820) extended toward the chromosomes and formed a loop that appears to have moved backward toward the centriole. This process repeated and formed many loops. The spindle microtubules appeared to directly implant or connect without interruption with the short-arm telomeric chromosome ends, nearest the polar area. Letter to the Editor 337 Fig. 4. Schematic reconstruction of Figures 1 to 3 and other high magnification electron micrographs (not shown) illustrating the interaction between chromosomes X and 4, and the mitotic spindle apparatus that consists of two parallel microtubules that run longitudinally between centriole and chromosomes forming several loops, which may function as a connecting hook that implants into the telomeric ends of the chromosome short arms. The centromere of chromosome 4, as indicated in this figure, was identified in another serial section (not shown) of the same number 4 chromosome shown in Figures 1 and 2. Note: p, short arm; q, long arm; c, centromere; cn, centriole; numbers 1 to 6 represent loops in the spindle apparatus (Figs. 2 and 3).