Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms.

Abstract

Studies of cell physiology and structure have identified many intriguing proteins that could be analyzed for function by using the power of yeast genetics. Unfortunately, identifying the homologous yeast gene with the two most commonly used approaches-DNA hybridization and antibody cross-reaction-is often difficult. We describe a strategy to identify yeast homologs based on protein function itself. This cloning-by-function strategy involves first identifying a yeast mutant that depends on a plasmid expressing a cloned foreign gene. The corresponding yeast gene is then cloned by complementation of the mutant defect. To detect plasmid dependence, the colony color assay of Koshland et al. [Koshland, D., Kent, J. C. & Hartwell, L. H. (1985) Cell 40,[393][394][395][396][397][398][399][400][401][402][403] is used. In this paper, we test the feasibility of this approach using the wellcharacterized system of DNA topoisomerase II in yeast. We show that (i) plasmid dependence is easily recognized; (ii) the screen efficiently isolates mutations in the desired gene; and (iii) the wild-type yeast homolog of the gene can be cloned by screening for reversal of the plasmid-dependent phenotype. We conclude that cloning by function can be used to isolate the yeast homologs of essential genes identified in other organisms.Many genes have been isolated that encode products essential for eukaryotic cells. The difficulty of genetic analysis in higher eukaryotes makes it attractive to study such genes by using the extraordinary power of yeast genetics. Unfortunately, because of the evolutionary distance between yeast and other experimental organisms, it is often difficult to identify the yeast homolog of a protein from another species by using the standard approaches of DNA hybridization and cross-species antibody reaction.Recent successes with substituting genes from other organisms for yeast genes suggest that an alternative approach will be very productive. Many examples are now available of foreign cDNAs encoding products that substitute fully for yeast proteins inactivated by mutation. For example, the human homolog of a Schizosaccharomyces pombe mitotic control gene, cdc2, was cloned by introducing a human cDNA library into a cdc2'5 strain and then screening for growth at the restrictive temperature (1). Additional examples of such conservation of function through evolution include other cell cycle proteins (2), protein transport components (3, 4), cytochrome c (5), and transcription factors (6, 7). The properties of the proteins in these examples suggest that not only monomer function, but also protein-protein interactions, have been conserved in evolution. The case of cytochrome c is particularly striking, not only because of the complex processing pathway that must be followed to produce a completely functional protein, but also because antisera raised against yeast or mouse cytochrome c do not react with the alternative cytochrome c; furthermore, DNA probes from yeast or rat cytochrome c genes do not cross-hybridize ...

Dual function of the tRNA(m5U54)methyltransferase in tRNA maturation

“…Strains with a mutation in the sup61 + gene required the TRM2 gene for growth A strain deleted for the TRM2 gene is viable and exhibits no apparent phenotype (Nordlund et al+, 2000)+ To elucidate the function of the Trm2 protein and/or the m 5 U 54 nucleoside in tRNA, a synthetic (synergistic) lethal screen was performed employing a red/white colony color assay (Kranz & Holm, 1990;Bender & Pringle, 1991)+ Synergistic lethality provides genetic evidence that two components physically interact or have overlapping functions+ Briefly, a haploid ura3 ade2 ade3 trm2 strain carrying a plasmid with the ADE3, URA3, and TRM2 genes was mutagenized and colonies were screened for the inability to lose the plasmid that is scored as uniformly red (nonsectored) colonies+ From 90,000 colonies, 21 strains with recessive mutations were identified that required the TRM2 gene for survival (see Materials and Methods)+ The mutants fell into several complementation groups of which one with four mutants was studied further+ The nonsectoring phenotype of these four strains was linked to a temperaturesensitive (Ts) growth phenotype+ Using a yeast genomic library, we identified three separate plasmids with identical inserts, which fully complemented the Ts and nonsectoring phenotypes+ Partial DNA sequencing of the insert from one of these plasmids and subsequent subcloning revealed that the sup61 ϩ gene restored sectoring at 30 8C and growth at 37 8C+ We also isolated plasmids carrying the eEF1A (TEF2) and the seryl-tRNA synthetase (SES1) genes that were able to partially complement the mutants+ Apparently, the TEF2 and SES1 genes were acting as dosage suppressors of the mutant phenotype (our unpubl+ data)+ The sup61 ϩ gene is a single copy and essential gene encoding tRNA CGA Ser , which is the only serine isoacceptor that decodes UCG codons (Etcheverry et al+, 1982)+ To confirm that sup61 ϩ was mutated, we cloned and sequenced the gene from the four mutant strains+ This analysis revealed that each strain contained a different single nucleotide exchange in the sup61 gene and the alleles were designated sup61-T51C, sup61-T44G, sup61-T47:2C, and sup61-T20A (Fig+ 1A)+ To unambiguously demonstrate that the various sup61 alleles were responsible for the dependency on TRM2, the wild-type sup61 ϩ gene was replaced with respective mutant allele in a trm2 strain carrying TRM2 on a plasmid (see Materials and Methods)+ The resulting strains (UMY2244 to 2247) exhibited a Ts growth phenotype and were dependent on the TRM2 plasmid for growth at 30 8C+ At 25 8C, the strains were able to spontaneously lose the TRM2 plasmid and generate the double mutant strains+ These double mutants did not to grow (sup61-T44G trm2, sup61-T20A trm2) or grew very slowly (sup61-T51C trm2, sup61-T47:2C trm2) at 30 8C, whereas the single trm2 and sup61 strains were able to grow (Fig+ 1B)+ Thus, the m 5 U modification or the Trm2 protein is important for growth in strains with mutant forms of tRNA CGA Ser + The Trm2 gene product influenced the relative level of mature and pre-tRNA CGA Ser in the sup61 mutants To establish the molecular mechanism for the synergistic effect of the trm2 null and the various sup61 alleles, the relative amount of tRNA CGA Ser was determined using northern blot analysis+ Wild-type, single, and double mutant strains were grown exponentially at 25 8C and shifted to 30 8C for 4 h, and total RNA was prepared from samples taken at respective temperature+ Because the sup61 gene contains an intervening sequence (Etcheverry et al+, 1979), mature and pre-tRNA CGA Ser was detected by using two different 32 Plabeled oligonucleotides as probes+ One probe was complementary ...…”

“…The screen used to isolate mutants was based on colony sectoring as described previously (Kranz & Holm, 1990;Bender & Pringle, 1991)+ Strain UMY1917, carrying plasmid pMJ1223 bearing the TRM2, ADE3, and URA3 genes, was grown in selective medium to approximately 2 ϫ 10 7 cells/ mL+ The cells were diluted with water, plated on YEPD plates, mutagenized with UV irradiation to approximately 10% survival, and incubated at either 25 or 30 8C+ Uniformly red colonies were restreaked at least twice and only those strains that continued to give nonsectored colonies were studied further+ The candidate strains were transformed with pMJ1274 and strains that regained the ability to sector were crossed to UMY2097 to test for dominance/recessiveness and for 2:2 segregation of the nonsectoring phenotype+ A low copy genomic library in YCp50 (Rose et al+, 1987) was transformed into strain UMY2108 (sup61-T44G trm2 pMJ1005) and transformants were selected on media lacking uracil+ Plasmids from transformants that regained the ability 332 M.J.O. Johansson and A.S. Byström to grow at 37 8C and lose the TRM2 plasmid at 30 8C were isolated and retransformed and those that stayed positive were partially sequenced and subjected to homology searches against the S. cerevisiae genome+…”

Evidence for a role of Sky1p-mediated phosphorylation in 3′ splice site recognition involving both Prp8 and Prp17/Slu4

“…Because SKY1 is not essential for growth (Siebel et al+, 1999), a synthetic lethal screen was conducted to identify genes that genetically interact with the kinase using the sectoring strategy described by Holm and coworkers (Kranz & Holm, 1990)+ This screen exploits the accumulation of red pigment in yeast containing defective ade2 and wild-type ADE3+ As diagramed in Figure 1A, SKY1 was deleted from a yeast strain (CH1305) carrying defective chromosomal alleles of both ade2 and ade3+ The resulting SL-sky1⌬ strain was covered with wild-type SKY1 in a URA3 and ADE3-marked plasmid (pCH1675-SKY1)+ Because SKY1 is dispensable for growth, the plasmid is easily lost and cells without ADE3 become white+ A colony may appear white or sector red and white, depending on how early the plasmid is lost during growth+ Red (nonsectoring) colonies will appear if a second site mutation makes SKY1 essential and therefore prevents the growth of cells that lose the plasmid bearing the wild-type ADE3 and SKY1 genes+…”

“…Yeast strains used in the current study are listed in Table 1+ The strain used for the synthetic lethal screen (CH1305) was a gift from the Holm laboratory (Kranz & Holm, 1990)+ SKY1 was deleted from all strains by using a kanamycin-resistance expression unit flanked by SKY1 genomic sequence as previously described (Yeakley et al+, 1999)+ The plasmid pCH1675-SKY1 was constructed by inserting the BamHISal I fragment containing SKY1 from pRS316-SKY1 (Siebel et al+, 1999) into the BamHI-SmaI sites of pCH1675 (Kranz & Holm, 1990)+ Wild-type SKY1 was similarly subcloned into the LEU2-marked plasmid YCpLac111+ The mutant YCpLac111-sky1 K-M was constructed by exchanging the PshAI-Pfl MI fragment from YCpLac111-SKY1 with the corresponding fragment from pYES2-sky1 K-M (Siebel et al+, 1999)+ The prp8 null strains yJU71 and yJU75 used for allele specificity and splice site suppression analyses were gifts from the C+ Guthrie laboratory+ The slu4 [ySJ134 (prp17-1), ySJ136 (prp17⌬ )] and slu7 [yDFA7C (slu7-1)] mutant strains, and plasmids pSJ3 (wild-type SLU4 in YCp50) and pDF64 (wild-type SLU7 in YCp50) were also gifts from the C+ Guthrie laboratory+ The prp18 null strain DH120R and the plasmid pDH101 bearing wild-type PRP18 were gifts from the D+ Horowitz laboratory+ The prp16 (prp23-ts514) and prp22 (prp22-ts107) mutant strains were gifts from the J+ Abelson laboratory (Vijayraghavan et al+, 1989)+ The URA/CEN plasmids carrying PRP16 (pS32) and PRP22 (p360) were gifts from the B+ Schwer laboratory+ All wild-type and mutant PRP8 alleles listed in Figure 2 and all ACT-CUP1 reporters used in Figure 4 were kindly provided by the C+ Guthrie laboratory (Lesser & Guthrie, 1993;Umen & Guthrie, 1996;Collins & Guthrie, 1999)+ Synthetic lethal screen SKY1 was first disrupted in CH1305 and the resulting SLsky1⌬ strain was covered with pCH1675-SKY1 (CEN, ARS, URA3, ADE3 )+ UV mutagenesis conditions were chosen to produce 25-30% viability on YP galactose plates+ Nonsectoring cells were isolated from approximately 100,000 surviving colonies and then subjected to 5-FOA selection+ Three synthetic lethals (SL34, SL39, and SL44 ) were obtained and all showed ts growth at 37 8C+ SL34 and SL44 had high reversion rates on 5-FOA plates following long incubation+ In contrast, SL39 was stable and therefore chosen for further functional studies+ The SL39 gene was cloned by complementing the ts phenotype with the Heiter yeast genomic DNA library, and identified as PRP8 by partial sequencing+ The prp8-39 mutation was determined by gap repair (Guthrie & Fink, 1991)+ Briefly, a series of restriction fragments was prepared from pSE363-PRP8 (HIS3, CEN, ARS ) (a gift from the C+ Guthrie laboratory) and transformed into the SL39 strain+ All but the PshAI-SpeI fragment were able to correct the ts phenotype, indicating the SL39 mutation(s) resides in this region+ The PshAI-SpeI gapped pSE363-PRP8 was then transformed into the SL39 strain and the repaired plasmid was isolated+ Sequencing of the PshAI-SpeI region in the p...…”

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2

“…Escherichia coli DH5a was used for cloning and propagation of plasmids+ Yeast strains used and constructed in this study are listed in Table 1+ Construction of plasmid pJPG203 (CEN, ADE3, URA3, GAR1) is described by Venema et al+ (1997) and construction of plasmids pJPG67 (CEN, TRP1, GAR1) and pJPG65 (CEN, TRP1, gar1⌬GAR ) in Girard et al+ (1994)+ Construction of pJPG209 was done as follows: A blunt-ended Sal ISmaI fragment from YDpK (Berben et al+, 1991) containing the LYS2 gene was isolated and used to replace the Bgl II fragment containing the URA3 marker gene in pFL38 (Bonneaud et al+, 1991) yielding pJPG207+ A 1+95-kb EcoRI-Pst I fragment from pJPG65 (CEN, TRP1, gar1⌬GAR ), containing the 39UTR of GAR1, the gar1 allele deleted from the GAR domains' coding regions, and the 59UTR of GAR1 was inserted in EcoRI-Pst I-digested pJPG207, yielding pJPG209 (CEN, LYS2, gar1⌬GAR )+ A plasmid directing the production of a GFP-Rrp8p fusion protein was constructed as follows+ The RRP8 ORF was PCR amplified from pJPG235 (see below) using the following oligonucleotides: 59-CCCCCGGATCCTTATGCATATATTGTA TATATTATATC-39 and 59-CCCCCGGATCCGTTATCTTCT TTTATAAATACAGGGCTTC-39+ The BamHI-digested PCR product was cloned at the BamHI site of plasmid pNOPPATA1L obtained from K+ Hellmuth and E+ Hurt, producing plasmid pJPG309+ The Pst I-TEV-insert-Pst I fragment from this construct was inserted into the Pst I site of plasmid pNOPGFPA1L, obtained from K+ Hellmuth and E+ Hurt+ A 2+9-kb SpeI-XhoI fragment from this construct was treated with the Klenow fragment of DNA PolI and inserted into pFL46S (Bonneaud et al+, 1991) and cut by Ecl136-HindIII also blunt-ended by the same enzyme, giving plasmid pJPG310+ As a control for the localization experiments, the equivalent GFP fragment from pNOPGFPA1L was cloned as a BamHI-HindIII fragment in BamHI-HindIII-digested pFL46S, giving plasmid pJPG311+ To produce a vector directing expression of ZZ-Rrp8p, an SphI fragment from pJPG309 was used to replace the SphI fragment of pJPG310, yielding pJPG312+ To construct plasmid pEV1 (CEN, ARS, ADE3, URA3, RRP8 ϩ ), an EcoRIScaI 2+1-kb fragment containing the RRP8 ϩ allele was cloned in EcoRI-Ecl136II-digested pFL39 (Bonneaud et al+, 1991) and a 2+2-kb PvuII-Sal I RRP8 ϩ fragment from this plasmid was then inserted in NruI-Sal I-digested pCH1122 (Kranz & Holm, 1990)+ Yeast cell transformation was performed according to Gietz et al+ (1995), with minor modifications: 6% DMSO is added prior to the heat shock and the final cell pellet is resuspended in 0+15 M NaCl+…”