Abstract
In preparations of highly condensed metaphase chromosomes, it is often difficult to resolve in situ hybridization signals of probes to nearby regions. This is in part, a result of traditional fixation procedures that use alcohol, acetic acid, and/or relatively strong ionic conditions, all of which can cause chromosome condensation. The limit of resolution may be improved by using free chromatin or DNA-halo preparations as described in Chapters 10 and 11 of this volume. An alternative method of detecting proximal DNA sequences is to use whole-mount, surface spread meiotic prophase chromosomes (1, 2), which have less condensed chromatin and a well defined axial core that is much longer than that of a metaphase chromosome. We have tested this system using two probes (3) against low-copy repeats, 5.5 map units apart on the X chromosome (4) as shown in Fig. 1. On metaphase chromosomes, these signals wereclose together and often overlapped. However, a distance of several micrometers separated the signals on pachytene chromosomes. This type of mapping is useful provided one keeps in mind that the chromatin is arranged in loops around a protein core, the synaptonemal complex (SC) (Fig. 2) (5), and that these loops are of considerable size relative to those of the metaphase chromosome. Thus, a sequence at the top of a 5-μm loop could appear some distance from the SC loop attachment point, depending on how the loop settles during chromosome preparation. Therefore, the size of the loop limits the resolution of the probes a few μm apart. However, if the probes are very close together (a few megabases), the decondensed chromatin permits resolving signals from probes to adjacent loci or to cosmid probes of different regions within the same gene.
References
Counce, S. J and Meyer, G. F (1973) Differentiation of the synaptonemal complex and the kinetochore in Locusta spermatocytes studied by whole-mount electron microscopy Chromosoma 44, 231–253.
Dresser, M. E. and Moses, M. J (1980) Synaptonemal complex karyotyping in spermatocytes of the Chinese hamster (Cricetulus griseus) IV. Light and electron microscopy of synapsis and nucleolar development by silver staining Chromosoma 76, 1–22.
Disteche, C M., Trantravahi, U., Gandy, S, Eisenhard, M, Adler, D., and Kunkel, L M (1985) Isolation and characterization of two repetitive DNA fragments located near the centromere of the mouse X chromosome Cytogen Cell Genet 39, 262–268
Moens, P. B and Pearlman, R. E. (1990) In situ DNA sequence mapping with surface-spread mouse pachytene chromosomes Cytogenet. Cell Genet 53, 219,220
Moens, P. B and Pearlman, R E (1989) Satellite DNA I in chromatin loops of rat pachytene chromatin and in spermatids Chromosoma 98, 287–294
Moens, P B and Pearlman, R E (1991) DNA sequence localization in meiotic chromosomes, in Methods in Cell Biology, vol 35 (Hamkalo, B. A and Elgin, S. C R, eds), Academic, New York, pp 101–108
Sambrook, J., Fritsch, E. F, and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 1–102, 6.3–6 35
Heery, D M., Gannon, F., and Powell, R (1990) A simple method for subcloning DNA fragments from gel slice. Trends Genet. 6, 173.
Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity Anal. Biochem 132, 6–13.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1994 Humana Press Inc.
About this protocol
Cite this protocol
Spyropoulos, B., Moens, P.B. (1994). In Situ Hybridization of Meiotic Prophase Chromosomes. In: Choo, K.H.A. (eds) In Situ Hybridization Protocols. Methods in Molecular Biology™, vol 33. Humana Press. https://doi.org/10.1385/0-89603-280-9:131
Download citation
DOI: https://doi.org/10.1385/0-89603-280-9:131
Publisher Name: Humana Press
Print ISBN: 978-0-89603-280-4
Online ISBN: 978-1-59259-520-4
eBook Packages: Springer Protocols