Construction of a large-insert yeast artificial chromosome library of the rat genome

Construction of a large-insert yeast artificial chromosome library of the rat genome Mammalian Genome 8, 284 (1997). 9 New York Inc. 1997 Construction of a large-insert yeast artificial chromosome library of the rat genome M.L. Haldi, 1 P. Lim, 1 K. Kaphingst, 1 U. Akella, 1 J. Whang, 1 E.S. Lander 1'2 lWhitehead/MIT Center for Genome Research, Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA 2Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA Received: 25 September 1996 / Accepted: 5 November 1996 The rat is the animal model of choice for the study of many aspects 100 , -4 of mammalian physiology. However, the rat has lagged behind the 90 ~ Mean size = 820 Median size = 815 mouse in terms of genetic studies, in large part owing to the lack 80 1 N=631 of appropriate genetic and genomic mapping resources. As part of our ongoing effort to provide mapping resources to the mammalian 70-~ genetics community, we have constructed the first yeast artificial chromosome (YAC) library of the rat genome. This library con- sists of approximately 41,500 clones with an average insert size of 820 kb and provides roughly 11-fold coverage. ~ 40 The methods used for preparation of the library are described by Haldi and colleagues (1996). Genomic DNA was prepared from kidney nuclei of Fischer 344 female rats. This DNA was ligated to pRML1 and pRML2 vector arms (Spencer et al. 1993). The liga- tion product was transformed into the yeast host strain J57D (MATer, ura3 trpl ade2-101 canl-lO0 leu2-3, 112 his3-6). The quality of the library was monitored during construction in two ways. Initially, the presence of rat DNA in the transformants was YAC size, kb assessed by a rapid PCR assay to detect the rat B2 repeat element. Fig. L Histogram showing the sizes of 631 randomly selected YAC This element is highly homologous to the murine B2 (denDunnen clones. and Schoenmakers 1987). The assay and primers used are de- scribed by Kusumi and associates (1993) and Haldi and colleagues excellent technical assistance and Maria Trolliet and Howard Jacob for (1996). Five percent of the transformants from each ligation were providing us with rat kidneys. screened, and typically greater than 95% of the clones yielded a PCR product visible by agarose gel electrophoresis with ethidium bromide. References The insert sizes of the YACs were determined by pulsed field gel electrophoresis as described by Haldi and coworkers (1996). den Dunnen JT, Schoenmakers JGG (1987) Consensus sequences of the Approximately 1.5% of the YACs from each ligation were exam- Rattus norvegicus B1- and B2 repeats. Nucleic Acids Res 15, 2772 ined. Figure 1 shows the distribution of YAC sizes in the 631 Haldi M, Strickland C, Lim P, VanBerkel V, Chen X-N, Noya D, Koren- clones tested. The mean size is 820 kb, and the median size in 815 berg J, Husain Z, Miller J, Lander E (1996) A comprehensive large- kb. Based on the estimated length of the rat genome as 3 billion bp, insert yeast artificial chromosome library for physical mapping of the this library provides 11-fold coverage. Were there not systematic mouse genome. Mamm Genome, 7, 767-769 cloning biases in YAC libraries, this would correspond to a prob- Hudson T, Stein L, Gerety S, Ma J, Castle A, Silva J, Slonim D, Baptista ability of 99.995% of a given locus being represented in the li- R, Kruglyak L, Xu S, Hu Z, Colbert A, Rosenberg C, Reeve-Daly M, brary. Thus, any gaps in coverage are likely to be due to cloning Rozen S, Hui L, Wu Z, Vestergaard C, Wilson K, Bae J, Maitra S, biases. Our experiences with human YAC libraries (Hudson et al. Ganiatsas S, Evans C, DeAngelis M, Ingalls K, Nahf R, Horton L, 1995) suggest the actual coverage is likely to be 98% of the ge- Anderson M, Collymore A, Ye W, Kouymoumjian V, Zemsteva I, Tan J, Devine R, Courtney D, Renaud M, Nguyen H, O'Connor T, Fizames home. C, Faure S, Gyapay G, Dib C, Morissette J, Odin J, Birren B, Goodman To ensure broad access by the mammalian genetics commu- N, Weissenbach J, Hawkins T, Foote S, Page D, Lander E (1995) An nity, the entire rat YAC library has been deposited with Research STS-based map of the human genome. Science 270, 1945-1954 Genetics, Inc. (Huntsville, Ala.), which will provide distribution Kusumi K, Smith J, Segre J, Koos D, Lander E (1993) Construction of a services. large-insert yeast artificial chromosome library of the mouse genome. Mamm Genome 4, 391-392 Acknowledgments. We thank Ndubuisi Azubuine and Rebecca Riley for Spencer F, Ketner G, Connelly C, Hieter P (1993) Targeted recombination- based cloning and manipulation of large DNA segments in yeast. Meth- ods: companion to Methods Enzymol 5, 161-175 Correspondence to: E.S. Lander or M.L. Haldi Springer-Verlag Mammalian Genome Springer Journals

Construction of a large-insert yeast artificial chromosome library of the rat genome

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Life Sciences; Cell Biology; Anatomy; Zoology
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