• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Feb 1986; 83(3): 730–734.
PMCID: PMC322938

Rapid mapping of antigenic coding regions and constructing insertion mutations in yeast genes by mini-Tn10 "transplason" mutagenesis.

Abstract

A "transplason" mutagenesis procedure was developed for the dual purposes of low resolution mapping of antigenic coding regions (using transposons) and constructing insertion mutations in yeast genes (by transplacement). Mini-Tn10 transposon derivatives containing both Escherichia coli and yeast selectable markers have been constructed. These elements are used to mutagenize lambda gt11 clones that express foreign antigens in E. coli. The transposition events are first selected in E. coli, and the effect of these insertions on antigen expression is used to locate the antigenic coding regions on the cloned DNA. Insertion mutations located within a desired yeast sequence are then substituted for the genomic copies by one-step gene transplacement. This provides a powerful method for rapidly mapping antigenic coding sequences of cloned genes and inactivating these genes in yeast to help determine their function. Several examples using this technique are presented.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Shortle D, Botstein D. Directed mutagenesis with sodium bisulfite. Methods Enzymol. 1983;100:457–468. [PubMed]
  • Goto T, Wang JC. Yeast DNA topoisomerase II is encoded by a single-copy, essential gene. Cell. 1984 Apr;36(4):1073–1080. [PubMed]
  • Johnson LM, Snyder M, Chang LM, Davis RW, Campbell JL. Isolation of the gene encoding yeast DNA polymerase I. Cell. 1985 Nov;43(1):369–377. [PubMed]
  • Young RA, Davis RW. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. [PMC free article] [PubMed]
  • Young RA, Davis RW. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. [PubMed]
  • Young RA, Bloom BR, Grosskinsky CM, Ivanyi J, Thomas D, Davis RW. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA. Proc Natl Acad Sci U S A. 1985 May;82(9):2583–2587. [PMC free article] [PubMed]
  • Ozkaynak E, Finley D, Varshavsky A. The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein. Nature. 1984 Dec 13;312(5995):663–666. [PubMed]
  • Frischauf AM, Lehrach H, Poustka A, Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. [PubMed]
  • Seed B. Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo. Nucleic Acids Res. 1983 Apr 25;11(8):2427–2445. [PMC free article] [PubMed]
  • Maurer R, Osmond BC, Shekhtman E, Wong A, Botstein D. Functional interchangeability of DNA replication genes in Salmonella typhimurium and Escherichia coli demonstrated by a general complementation procedure. Genetics. 1984 Sep;108(1):1–23. [PMC free article] [PubMed]
  • Foster TJ, Davis MA, Roberts DE, Takeshita K, Kleckner N. Genetic organization of transposon Tn10. Cell. 1981 Jan;23(1):201–213. [PubMed]
  • Way JC, Davis MA, Morisato D, Roberts DE, Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984 Dec;32(3):369–379. [PubMed]
  • Elledge SJ, Walker GC. Phasmid vectors for identification of genes by complementation of Escherichia coli mutants. J Bacteriol. 1985 May;162(2):777–783. [PMC free article] [PubMed]
  • Botstein D, Falco SC, Stewart SE, Brennan M, Scherer S, Stinchcomb DT, Struhl K, Davis RW. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. [PubMed]
  • Jorgensen RA, Rothstein SJ, Reznikoff WS. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. [PubMed]
  • Scherer S, Davis RW. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. [PMC free article] [PubMed]
  • Rothstein RJ. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. [PubMed]
  • Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. [PMC free article] [PubMed]
  • Hieter P, Mann C, Snyder M, Davis RW. Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss. Cell. 1985 Feb;40(2):381–392. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...