Genetics, Vol. 181, 837-838, March 2009, Copyright © 2009
doi:10.1534/genetics.109.100586

The 2009 Novitski Prize

James E. Haber

THE Novitski Prize, established in 2008 by the family of the remarkable geneticist Ed Novitski, is awarded for innovative experimental approaches and extraordinary creativity in solving a significant problem in genetics research. The 2009 Novitski Prize honors two pioneers of gene targeting, Rodney Rothstein of Columbia University and Kent Golic of the University of Utah. The development of transformation in budding yeast by HINNEN et al. (1978) made it possible to clone genes by complementation and raised the prospect that one could also delete or mutate genes selectively. Rod Rothstein contributed two fundamental ideas to our current ability to knock out genes and to knock in specific mutations. First, in collaboration with Terry Orr-Weaver and Jack Szostak, Rothstein demonstrated that a double-strand break within a yeast gene sequence on a plasmid dramatically increases the likelihood of the plasmid integrating at the homologous site on a chromosome (ORR-WEAVER et al. 1981, 1983). These experiments provided much of the impetus for developing a comprehensive double-strand-break-repair model of recombination (SZOSTAK et al. 1983). But Rothstein's interest was also focused on methods to create specific mutations in yeast. The earliest strategy to knock out a gene was to integrate a mutated gene sequence on a plasmid into the chromosome, creating a duplication, and then to select or screen for excision events that reversed the integration but left the mutant sequence behind (SCHERER and DAVIS 1979). An alternative strategy was to integrate a segment of the gene lacking its 5'- and 3'-ends, thus creating two tandem and truncated copies of the gene (SHORTLE et al. 1982). Being able to direct the integration by linearizing the plasmid with a restriction endonuclease made these approaches far more efficient (ORR-WEAVER et al. 1981). But it was Rothstein's "ends-out" transformation procedure, modestly published in Methods in Enzymology (ROTHSTEIN 1983), that revolutionized yeast gene targeting (Figure 1A). Exactly how the transformed linearized DNA replaces homologous sequences on a chromosome is not yet fully understood, even if one liberates a fragment inside the nucleus from another site within the genome (LEUNG et al. 1997; NEGRITTO et al. 1997; LANGSTON and SYMINGTON 2004); but in budding yeast, gene replacement is a surprisingly accurate process. The ease with which deletions and targeted mutations could be accomplished made it possible to create collections of deletions of every open reading frame in budding yeast, along with ways to insert epitope tags, GFP fusions, and temperature-sensitive degrons. Rothstein's simple idea was later extended to gene knock-out technology in mammalian cells, earning Oliver Smithies and Mario Cappecchi the 2007 Nobel Prize in Physiology and Medicine.


Figure 1
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FIGURE 1.—

Ends-out gene targeting. (A) In budding yeast, a linearized DNA fragment containing a selectable marker in place of an open reading frame (ORF) recombines with homologous 5' and 3' sequences to create a full gene deletion. (B) In Drosophila, to create a targeted gene mutation requires several steps: (i) A modified P element (shaded box) is created containing FRT sites (solid arrows) surrounding 3' and 5' homologous sequences that flank all or part of the ORF. The homologous sequences are separated by an 18-bp I-SceI endonuclease recognition site (inverted triangles). (ii) Flies containing the P element are crossed with flies containing constructs that express both the site-specific FLP recombinase and the I-SceI endonuclease. FLP first pops out a circular DNA fragment and then I-SceI linearizes the excised segment. (iii) The linear fragment has an "ends-out" configuration that can replace part of the ORF with other sequences.

 
Despite these advances in yeast and in mammalian cells, gene targeting in Drosophila remained elusive, even a decade after yeast targeting had become routine. SPRADLING and RUBIN (1982) showed that P elements could be mobilized to create germline mutations but attempts to transform the germline with linearized DNA fragments carrying a selectable marker failed. Kent Golic had succeeded in expressing the yeast site-specific recombinase FLP in flies, allowing it to direct reciprocal exchanges between two FRT sites carried in P elements (GOLIC 1991), a technique with profound implications for mosaic analysis in Drosophila. His studies of this system in the germline had also established that one could create chromosome translocations and other alterations by directing reciprocal exchanges between two FRT sites carried in P elements (GOLIC 1991) at different locations in the genome. To improve gene targeting, Golic realized it would be necessary to preestablish a stable version of a fragment that one wanted to use in transformation and then liberate it in the cell. This trick had already been demonstrated in budding yeast, using the rare-cutting HO and I-SceI endonucleases. Working primarily with his postdoc, Yikang Rong, Golic devised a powerful strategy to direct gene knockouts in the fly germline (RONG and GOLIC 2000). The strategy involved the use of both FLP and I-SceI (Figure 1B). First, the 5' and 3' regions of the gene to be knocked out were placed, but in reverse orientation (3' then 5'), within a pair of FRT sites in a P element that could be introduced at a random site into the Drosophila genome. In a subsequent cross, the P element was introduced into the presence of two constructs expressing FLP and the I-SceI endonuclease. FLP resulted in the popping out of the gene-targeting region from the P element as a circular fragment. Cleavage of the circular DNA resulted in a linear fragment that could recombine in an ends-out fashion to delete part of the target gene. And it worked! Subsequently Golic and his colleagues developed "ends-in" strategies to disrupt genes and to knock in specific mutations (RONG et al. 2002).

Ed Novitski himself was a master in chromosome mechanics (CROW et al. 2006), and therefore it is most appropriate to award this year's GSA Novitski Prize to these two inventive recipients.
Figure 2
Rodney Rothstein


Figure 3
Kent Golic

LITERATURE CITED

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HINNEN, A., J. B. HICKS and G. R. FINK, 1978 Transformation of yeast. Proc. Natl. Acad. Sci. USA 75: 1929–1933.[Abstract/Free Full Text]

LANGSTON, L. D., and L. S. SYMINGTON, 2004 Gene targeting in yeast is initiated by two independent strand invasions. Proc. Natl. Acad. Sci. USA 101: 15392–15397.[Abstract/Free Full Text]

LEUNG, W., A. MALKOVA and J. E. HABER, 1997 Gene targeting by linear duplex DNA frequently occurs by assimilation of a single strand that is subject to preferential mismatch correction. Proc. Natl. Acad. Sci. USA 94: 6851–6856.[Abstract/Free Full Text]

NEGRITTO, M. T., X. WU, T. KUO, S. CHU and A. M. BAILIS, 1997 Influence of DNA sequence identity on efficiency of targeted gene replacement. Mol. Cell. Biol. 17: 278–286.[Abstract/Free Full Text]

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SCHERER, S., and R. W. DAVIS, 1979 Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc. Natl. Acad. Sci. USA 76: 4951–4955.[Abstract/Free Full Text]

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SPRADLING, A. C., and G. M. RUBIN, 1982 Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218: 341–347.[Abstract/Free Full Text]

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