A Method to Generate Full-Length cDNA Molecules from Yeast Two-Hybrid Clones and RACE Products

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A Method to Generate Full-Length cDNAMolecules from Yeast Two-HybridClones and RACE Products

Charles S. Hemenway, Benjamin W. Halligan, andGrahame C. D. GouldDepartment of Pediatrics and the Tulane Cancer Center,Tulane University School of Medicine, New Orleans,Louisiana 70112

Received September 10, 1998

The utility of the yeast two-hybrid system in identi-fying proteins that physically interact is well estab-lished (1). Widespread application of the technique hasbeen facilitated by the large number of two-hybridcDNA libraries available from individual laboratories,tissue banks, and commercial sources. Many two-hy-brid libraries containcDNA inserts with an averagesize of ,1.5 kb. The relatively small size of the cDNAinserts has several advantages. Some large fusion pro-teins are poorly targeted to theyeast nucleus, thuspreventing transactivation of the reporter gene(s) (2).In addition, other large fusion proteins presumablyadopt a higher order structure that prevents interac-tion with true binding partners. Thus, the inclusion ofsmall cDNA molecules in two-hybrid libraries may im-prove the sensitivity of the selection process.

Implicit in the design of these libraries is that two-hybrid clones frequently contain a partial, rather thanfull-length, cDNA molecule. Full-length cDNA canthen be cloned by probing a second cDNA library withthe partial cDNA fragment. A less time-consumingapproach is the rapid amplification of cDNA ends(RACE),1 in which the complete 59 and/or 39 ends of thecDNA molecule are amplified by PCR using oligonucleo-tide primers derived from the partial cDNA sequence(3). While RACE provides valuable sequence informa-tion, the technique generates another partial cDNAmolecule. Therefore, to produce a full-length cDNA,

oligonucleotide primers can be designed to amplify thecomplete molecule from the original RACE library orsome other source of cDNA. This may not be techni-cally feasible in all cases, however. As an alternative,the RACE and two-hybrid cDNA fragments can beligated at an overlapping restriction endonuclease site.Unfortunately, this is often not possible, particularly ifthe “overlap” is small, thus limiting the number ofsuitable endonuclease sites.

We have found that complete cDNA molecules can beproduced quickly and reliably from overlapping two-hybrid and RACE clones through homologous recombi-nation in yeast. This method generates a functionalyeast two-hybrid plasmid that contains the full-lengthcDNA molecule as well as restriction endonucleasesites that facilitate the direct cloning of the moleculeinto other plasmid vectors. PCR amplification of theRACE cDNA fragment is required but is readily accom-plished as purified DNA is used as a template for thereaction. Therefore, we offer a useful alternative toproducing full-length cDNA molecules from yeast two-hybrid clones and RACE products that further exploitsthe genetic properties of Saccharomyces cerevisiae. Themethod is based on plasmid construction by homolo-gous recombination in yeast, first described by Botsteinand frequently referred to as “gap repair” (4). PCR-based techniques of gene construction by recombina-tion in yeast have wide applicability and utility (5–7).We have modified previously described methods to fitthe specific circumstances encountered with a two-hybrid partial cDNA clone described above.

Recently, we have employed the yeast two-hybridsystem to clone two mouse Polycomb group (PcG) genes(8). In each case, the two-hybrid plasmid that wasisolated contained a cDNA molecule fused, in-frame, tothe GAL4 activation domain but contained no apparenttranslation initiation sequence and lacked the 59 cod-ing region of homologous PcG genes. To determine thecomplete coding sequences, the 59 ends of the cDNAswere isolated by RACE. This was accomplished using aPcG sequence-specific antisense oligonucleotide (“oligo-nucleotide B”), an adaptor sequence primer, and a com-mercially obtained adaptor-ligated cDNA library(Clontech). The 59 ends were amplified by PCR, clonedin the pGEM-T vector (Promega), and sequenced.

Next, we sought to produce full-length cDNA moleculesfrom the 59 RACE products and two-hybrid clones. DNAfragments to generate complete cDNAs by gap repair ofthe two-hybrid clones were produced as follows (Fig. 1):Oligonucleotide A (sense) contains 33 bases identical tothe pGAD10 vector sequence extending from the end ofthe GAL4 activation domain sequence across the vectorpolylinker sequence. The oligonucleotide continues withan additional 21 bases identical to the 59 sequence of theRACE product and includes the translation initiationcodon. The heterologous sequences are joined such that1 Abbreviation used: RACE, rapid amplification cDNA ends.

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Analytical Biochemistry 268, 161–162 (1999)Article ID abio.1998.30340003-2697/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

the GAL4 activation domain coding sequence continues,in-frame, with the PcG gene coding sequence. Oligonu-cleotide B (antisense) corresponds to the 39 end of theRACE product and was originally designed as the gene-specific primer for the 59 RACE reaction. With the cloned59 RACE product as a template, oligonucleotide primersA and B were used to PCR amplify a DNA fragmentusing Pfu polymerase in a reaction volume of 50 ml. Theresulting DNA molecule contains a 59 end with homologyto the GAL4 activation domain sequence and a 39 endwith homology to the partial cDNA contained in the two-hybrid vector.

Next, 1.0 mg of the two-hybrid plasmid was cut at aunique site in the multicloning sequence 59 to thecDNA insert, and both the PCR product and linearizedplasmid DNA were gel purified. Purified DNA was thenused to transform yeast strain PJ69-4A by the stan-dard LiOAc method of Gietz (9). In two independentprocedures, each employing a different PcG gene, 50 mlof competent yeast cells yielded 50–100 Leu1 colonies.Individual Leu1 yeast colonies were selected fromwhich plasmid DNA was isolated. Approximately 50%of the plasmids contained a full-length cDNA molecule,while the others contained the partial cDNA, presum-ably due to religation of the original cut plasmid.

The resulting plasmid vectors contain the completePcG gene coding sequences fused, in-frame, to theGAL4 activation domain sequence in the parent vectorpGAD10. A portion of the pGAD10 59 multicloning siteis preserved and the cDNA can therefore be easilysubcloned in other vectors. The process of generating acomplete cDNA molecule from a partial two-hybridclone and a 59 RACE cDNA can be completed in 1 week.

The most important requirement is the appropriatedesign of an oligonucleotide primer containing 59 se-quence that will correctly target the PCR-generated 59cDNA molecule (primer A). Several factors should beconsidered in its design. First, the 59 end of primer Ashould be identical to the intended plasmid integrationsite. The efficiency of homologous recombination of thePCR-generated cDNA fragment is determined, in part,by the length of this sequence. Previous studies haveshown that 30 bp of perfect homology at each end of themolecule is sufficient for targeted integration (10). Sec-ond, the 39 end of primer A must be designed to success-fully PCR-amplify the cloned 59 RACE cDNA. Finally, ifdesired, additional restriction endonuclease sites can beincorporated into the mid-portion of the oligonucleotide;however, it should often be possible to take advantage ofthe multicloning sequence at the targeted integrationsite. Thus, primer A will typically be 50–60 nt in length.

In sum, this report describes a method of generatingfull-length cDNA molecules that exploits the efficiencyand specificity of homologous recombination in yeast.We believe that this technique may prove useful toinvestigators who have made use of the yeast two-hybrid system to identify as-yet uncharacterized genes.

Acknowledgments. The authors thank John Lewy for his supportand Laura Levy for valuable guidance. This work was supportedby a grant from the Cancer Association of Greater New Orleansto C.S.H.

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FIG. 1. Summary of the method for generating full-length cDNA.See text for details. A and B denote oligonucleotide primers.GAL4AD and MCS indicate the GAL4 transactivating domain andthe multicloning sequence of the two-hybrid vector, respectively. Thetranslation initiation start codon is indicated by atg. URE representsa unique restriction endonuclease site. The PCR-generated 59 frag-ment is targeted to the desired site and integrated into the plasmidvector by homologous recombination in yeast.

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