[
Methods Cell Biol,
1995]
The clone-based physical map of the 100-Mb Caenorhabditis elegans genome has evolved over a number of years. Although the detection of clone overlaps and construction of the map have of necessity been carried out centrally, it has been essentially a community project. Without the provision of cloned markers and relevant map information by the C. elegans community as a whole, the map would lack the genetic anchor points and coherent structure that make it a viable entity. Currently, the map consists of 13 mapped contigs totaling in excess of 95 Mb and 2 significant unmapped contigs totaling 1.3 Mb. Telomeric clones are not yet in place. The map carries 600 physically mapped loci, of which 262 have genetic map data. With one exception, the physical extents of the remaining gaps are not known. The exception is the remaining gap on linkage group (LG) II. This has been shown to be bridged by a 225-kb Sse83871 fragment. Because the clones constituting the map are a central resource, there is essentially no necessity for individuals to construct cosmid and yeast artificial chromosome (YAC) libraries. Consequently, such protocols are not included here. Similarly, protocols for clone fingerprinting, which forms the basis of the determination of cosmid overlaps and the mapping of clones received from outside sources and has to be a centralized operation, and YAC linkage are not give here. What follows is essentially a "user's guide" to the physical map. Details of map construction are given where required for interpretation of the map as distributed. The physical mapping has been a collaboration between the MRC Laboratory of Molecular Biology, Cambridge, United Kingdom (now at The Sanger Centre, Cambridge, UK) and Washington University School of Medicine, St. Louis, Missouri. Inquiries regarding map interpretation, information, and materials should be addressed to alan@sanger.ac.uk or rw@nematode.wustl.edu.
[
Methods Cell Biol,
1995]
Although Caenorhabditis elegans was originally chosen as a model organism for cell biology with serial section electron microscopy (EM) methods in mind, these methods have remained a daunting challenge. There is an apocryphal story that Nichol Thomson originally advised Sydney Brenner that C. elegans was unsuitable for electron microscopy and that Brenner should choose another species. Other experienced microscopists have probably shared similar dark thoughts from time to time. Nonetheless, the worm's very small size, simple organization, and cablelike nervous system have permitted Brenner's colleagues to characterize every cell and cell contact in the wild-type animal, potentiating the genetic characterization of cellular development in remarkable detail. We attempt to provide an adequate background for anyone to initiate EM studies of C. elegans. Two decades ago, as the first of Brenner's postdoctoral fellows left his laboratory to establish new worm laboratories, it was standard practice to include an EM component in their studies. Their combined efforts to characterize the adult animal's cell types and the essential steps in its development helped to erect a lovely scaffold of key manuscripts, capped by the description of the "Mind of the Worm" in some 600 micrographs and 175 drawings. Many of these works required technical heroics or suffered long delays before publication. Most people later chose to leave electron microscopy behind in pursuit of molecular quarry. The fruits of their molecular and genetic studies should soon stimulate a renewed flowering of electron microscopy. We hope to smooth your entry or reentry into these techniques. We also summarize our methods for three-dimensional (3D) image reconstruction, based largely on film techniques introduced by John White and Randle Ware. Digital imaging techniques seem poised to make 3D reconstruction more accessible, and may simplify the exchange of morphological data between laboratories. We discuss several computer systems that the C. elegans community could adopt for high-resolution studies of structure and function. In addition, we briefly cover several specialized specimen preparation techniques for electron microscopy, including freeze fracture and electron microscopic immunocytochemistry.