Sponsored by the U.S. Department of Energy Human Genome Program
Human Genome News Archive Edition
Human Genome News, July 1993; 5(2)
The NIH National Center for Human Genome Research supported a workshop January 29-31 in Herndon, Virginia, to discuss problems associated with constructing yeast artificial chromosome (YAC) libraries. Major advances in molecular genetics over the past few years can be attributed to the use of YAC cloning, and the availability of a number of large YAC libraries has facilitated genome research on humans as well as on the mouse and other model organisms. Despite these marked successes, a number of problems remain, among the most frustrating of which is the difficulty in constructing YAC libraries, even by experienced investigators.
The workshop was organized by Daniel Cohen (Centre d'Etude du Polymorphisme Humain/Genethon), Eric Green (Washington University), and Rodney Rothstein (Columbia University). The group of 25 investigators with expertise in large DNA cloning, yeast biology, and/or YAC cloning discussed experiences and shared protocols. Presentations and discussions were organized into sessions on (1) DNA purification and construction of ligation mixes, (2) yeast transformation, (3) YAC vectors and yeast hosts, and (4) plans for future studies.
Almost every aspect in the multistep construction of YAC clones was discussed in detail. In many cases, attendees agreed on a particular procedure or reagent, while markedly different experiences were reported for other steps. One important conclusion emerging from the workshop was that no single "consensus" protocol will provide large numbers of clones on a consistent basis. Attendees generally agreed that success in achieving consistent results requires a long, sustained commitment over many months and the willingness to perform numerous parallel controls to test different steps in the overall process.
An example of this commitment is the careful attention required in preparing and handling yeast spheroplasts. All investigators are using adaptations of the spheroplast-transformation protocol originally described by Peter Burgers (Washington University School of Medicine) in Analytical Biochemistry 163:391 (1987). At the workshop, Burgers presented unpublished data demonstrating the dramatic effect of slightly altered polyethylene glycol (PEG) concentrations on the transformation efficiency of large DNA molecules. His data indicated that titrating the optimal concentration for transformation is important for each new batch of PEG.
Simon Foote (Whitehead Institute) described his method for establishing and maintaining optimal spheroplasting in the construction of a YAC library enriched for DNA from the human Y chromosome. MaryKay McCormick (Los Alamos National Laboratory) discussed her use of frozen spheroplasts in making YAC clones.
Definitive solutions to problems of YAC chimerism and instability are not yet available, although several promising lines of investigation are under way. In the characterization of libraries constructed with recombination-deficient yeast strains, preliminary data is encouraging.
As a result of the workshop, an electronic newsgroup has been established to provide a forum for topics related to the preparation, analysis, and use of YACs. Information about alternative host strains, YAC stability, and problems with reagents used in YAC library construction will be transmitted rapidly to all subscribers. A diverse group of investigators using YACs is expected to participate in the newsgroup, which is distributed from the BIOSCI computer (net.bio.net on the Internet). Distribution may later include USENET if usage grows sufficiently.
E-mail subscription requests should be sent to firstname.lastname@example.org only; no special computer commands are necessary. Messages to be posted, announcements, and queries about YAC construction should be sent to email@example.com. Investigators constructing YAC libraries or using YAC clones are encouraged to participate in this electronic newsgroup for rapid submission of important unpublished information about YAC cloning.
[Eric Green, Washington University School of Medicine]
The electronic form of the newsletter may be cited in the following style:
Human Genome Program, U.S. Department of Energy, Human Genome News (v5n2).
The Human Genome Project (HGP) was an international 13-year effort, 1990 to 2003. Primary goals were to discover the complete set of human genes and make them accessible for further biological study, and determine the complete sequence of DNA bases in the human genome. See Timeline for more HGP history.
Published from 1989 until 2002, this newsletter facilitated HGP communication, helped prevent duplication of research effort, and informed persons interested in genome research.