Vol. 10, No. 11

MOLECULAR AND CELLULAR BIOLOGY, Nov. 1990, p. 6097-6100 0270-7306/90/116097-04$02.00/0

Functional Analysis of a Duplicated Linked Pair of Ribosomal Protein Genes in Saccharomyces cerevisiae DAVID M. DONOVAN, MARY P. REMINGTON, DAVID A. STEWART, JOSEPH C. CROUSE, DAVID J. MILES, AND NANCY J. PEARSON*

Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, Maryland 21228 Received 16 April 1990/Accepted 15 August 1990

Ribosomal protein genes RP28 and S16A (RP55) are closely linked. Another set of this pair of genes exists in the genome (copy 2), genetically unlinked to copy 1. By using gene replacement techniques, we have shown that RP28 from copy 1 is required for vegetative growth and that the cells need S16A from copy 2 to achieve maximum growth rate. Many ribosomal protein (RP) genes appear to be duplicated in the Saccharomyces cerevisiae genome, although others occur in single copy (9). Duplicated genes include RP51 (1), S10 (10), RBL16 (18), RP28 and RP55 (S16A) (13), and L2 (11). In all cases where sequences are known, the coding regions of the duplicate genes are highly homologous, suggesting that both copies are functional. In the case of RP51, it has been shown that the two gene copies, A and B, contribute about 60 and 40% to the RP51 mRNA pool, respectively (1). In the case of S16A-1 and S16A-2, it has been demonstrated that the gene products from these two copies appear in the ribosome at a ratio of about 1:2, respectively (16). Rotenberg et al. (18) have shown that the accumulation of RBL16A transcripts is about half that of RBL16B. The results of Lucioli et al. (11) demonstrate that the L2A and L2B RP genes contribute unequally to the steady-state levels of L2 mRNA, with L2A representing 72% and L2B producing 28% of the total L2 mRNA. Considering the fact that the synthesis of RPs appears to be coordinately regulated under many conditions (7, 8, 22), it is unclear why there should be two differentially expressed copies of some RP genes and one copy of others. In order to investigate this question, we have examined the consequences of removing a linked set of RP genes (RP28-S16A) from the genome and an unlinked duplicate copy of this set (17) in separate experiments. Replacement of RP28-S16A copy 1. Copy 1 of this linked pair of genes was replaced with the URA3 gene by standard methods, as described in reference 19. To do this, a plasmid was constructed that contained a 3.1-kb BamHI fragment with the URA3 gene, flanked by sequences that normally flank RP28-S16A copy 1 (Fig. 1A). This fragment was used to transform the diploid strain JL6B/JL6D (see Table 1 for genotype) by a method modified from Ito et al. (6, 20). Replacement of the chromosomal RP28-S16A copy 1 on one chromosomal homolog was confirmed by Southern analysis (21). The 6.8-kb BamHI fragment detected by Southern analysis as shown in Fig. 1A represents the parental BamHI fragment, and the 3.1-kb fragment in lanes 2 and 3 (transformed diploids) is the replacement BamHI fragment present on one homolog in the diploid. Genetic analysis of RP28-S16A copy 1 replacement. A total of 38 tetrads were dissected by standard methods (15) from *

the two diploids shown by Southern analysis to be heterozygous for a deletion of copy 1. Both produced tetrads with only one or two viable spores, compared with the parent diploid, which produces tetrads with three or four viable spores 75% of the time. Furthermore, all the spores that produced colonies in the deleted diploid were Ura-. This indicates that the replacement of copy 1 of this linked pair of genes inhibits vegetative growth. However, spores that did not form colonies did germinate and divide two to three times, as determined by examination of the dissection plates. Rescue of RP28-S16A copy 1 gene replacement. To determine whether the growth defect caused by the deletion of RP28-SJ6A copy 1 was brought about by deletion of RP28-1, SJ6A-1, or some flanking sequence that was removed, each individual gene (RP28-1, RP28-2, SJ6A-1, S16A-2) was introduced back into the deleted diploid on a replicating plasmid (see Fig. 3 for structure of the replicating plasmids). Diploids transformed with these plasmids in separate experiments were then sporulated and dissected. The results showed that of 49 viable haploid progeny that contained the deletion of copy 1 (Ura+) all contained a plasmid harboring either the RP28-1 or RP28-2 gene (Leu+). One can conclude from this analysis that it is the absence of RP28-1 that causes the lethality and not the absence of S16A-1 or flanking sequences, since RP28-2 has different flanking sequences but still complements the lack-of-growth phenotype. This experiment also demonstrates that RP28-2 is an active gene since it complements the deletion of RP28-1 when present on a high-copy-number plasmid. Furthermore, the results of these experiments are consistent with the fact that Herruer et al. (5) have estimated by primer extension analysis that at least a sixfold difference exists between the amounts of mRNA transcribed from the duplicate RP28 genes (RP28-2 being sixfold lower than RP28-1). Therefore, the RP28 gene pair represents an extreme case of differential expression between duplicate yeast RP genes such that the level of expression of one gene is not even sufficient to support growth. Replacement of RP28-SJ6A copy 2. The replacement of copy 2 of RP28-S16A was accomplished basically in the same manner as the replacement of copy 1, as diagrammed in Fig. 1B. A 2.5-kb EcoRI-ClaI fragment containing the TRPI gene flanked by sequences which normally flank RP28-SI6A copy 2 was used to transform the diploid strain YNN281/YNN282. Several Trp+ transformants were analyzed by Southern analysis. Those with the predicted South-

Corresponding author. 6097

6098

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FIG. 1. Replacement of RP28-S16A copy 1 by URA3 (A) and replacement of RP28-S16A copy 2 by TRP1 (B). Schematics of each replacement are shown on the left. See text for details. (A) Autoradiograph showing genomic blots in which the DNA was cut with BamHI and probed with the nick-translated 3.1-kb transforming fragment. Lane 1, JL6B/JL6D; lanes 2 and 3, DNA from two stable URA+ JL6B/JL6D transformants. (B) Autoradiograph showing genomic blots where the DNA was cut with EcoRI and probed with the 4.0-kb EcoRI fragment from the copy 2 genomic clone (see text for details). This fragment contains part of RP28-2 and the 5'-flanking region, shown schematically to the left. Lane 1, YNN281/282; lane 2, stable Trp+ transformant of YNN281/282; lanes 2A, 2B, 2C, and 2D, DNA isolated from the four haploids of tetrad no. 2 from a YNN281/282 Trp+ transformant no. 5. (2A and 2B had trp- large spore colonies [U = 2.5 to 3 h at 23°C in liquid YPD] and 2C and 2D produced Trp+ small spore colonies [U = -6 h at 23°C in YPD]). ern pattern consistent with a deletion of RP28-SJ6A copy 2

further analyzed (Fig. 1B). For this analysis, DNAs from the parent diploid and several transformants were cut with EcoRI and probed with a 4.0-kb EcoRI fragment from RP28-SJ6A copy 2, as shown in Fig. 1B. The 4.0-kb Southern band is the parental fragment, and the 2.5-kb band is predicted replacement fragment. were

Genetic analysis of RP28-S16A copy 2 gene replacement.

Three diploids that showed the expected Southern pattern

for a heterozygous gene replacement were sporulated and dissected. When spore colonies were examined on yeast peptone dextrose (YPD) dissection plates after three days at 23°C, a specific pattern could be observed for all three diploids. Four spore tetrads occurred frequently, and all segregated in a ratio of two small spore colonies to two large spore colonies (Fig. 2). On the basis of the analysis of 34 tetrads, all the small spore colonies were determined to be Trp+ (replacement marker) and all the large colonies were

TABLE 1. Strains used in this study and their genotypes

Genotype

Strain

Source

JL6B/JL6D

MATa cry'r leu2-3 leu2-112 ura3-5 trpl MATa CRYPS leu2-3 leu2-112 ura3-52 trpl

J. Woolford

YNN281/282

MATa trpl-A his3-A200 ura3-52 lys2-801a ade2-lo MATct trpl-A his3-A200 ura3-52 lys2-801a ade2-lo

YGSCa

JL6D/1A

MATa leu2-3 leu2-112 ura3-52 trp_ his3-A200 RP28-56A(2) MATa leu2-3 leu2-112 ura3-52 TRPI HIS3 RP28-S16A(2)::TRPI

This study

ClA-lB/lA

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This study

a YGSC, Yeast Genetic Stock Center.

NOTES

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FIG. 2. Illustration of the small and large colony phenotypes of tetrads dissected from a diploid that was heterozygous for the deletion of copy 2 (YNN281/282 Trp+ transformant no. 5).

found to be Trp-. We measured the growth rate in liquid YPD for eight spore progeny from two tetrads. The results were that the Trp+ spores had an average generation time in YPD at 23°C of 4 to 5 h, whereas the generation time of the Trp- haploids was in the range of 2 to 3 h. Therefore, the small colony size of the haploids harboring the replacement of copy 2 was caused by a slower cellular growth rate. Thus, although the deletion of copy 2 RP28-S16A was not lethal, it did produce an observable mutant phenotype. Rescue of the RP28-S16A copy 2 gene replacement. To determine exactly what element on the deleted segment of RP28-S16A copy 2 caused the slow-growth phenotype, the four RP gene-containing plasmids described previously and shown in Fig. 3 were transformed into the diploid that was heterozygous for the deletion of copy 2 in separate experiments. The transformants were then sporulated and dissected. On the basis of the analysis of 127 haploid progeny, the results showed that only the presence of S16A-1 or SJ6A-2 on a replicating plasmid could complement the E

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FIG. 4. Polysome analysis of tetrad no. 2 from diploid no. 5, heterozygous for copy 2 replacement. Polysome gradients (7 to 47%) were prepared according Warner et al. (23). The top of the gradients are to the left. 40S, 60S, and 80S peaks are indicated. (2A and 2B have an intact copy 2, whereas 2C and 2D have copy 2 replaced with TRPI.)

small-colony phenotype of haploids deleted for copy 2. Thus, the deletion of SJ6A-2, not RP28-2 or flanking sequences, is responsible for the slow-growth, small-sporecolony phenotype. Polysome analysis of copy 2 gene replacement. Because cells deleted for copy 2 are viable, we could examine the effect of this deletion on general protein synthesis by analyzing polysome profiles. Figure 4 diagrams four polysome profiles from the members of a tetrad dissected from a diploid that is heterozygous for this replacement (YNN281/ 282 copy 2 AI+). The results show (i) that the amounts of 40S subunits are significantly reduced compared with the amounts of 60S subunits in haploid segregants that are deleted for RP28-SJ6A (copy 2) and compared with the two sister spore progeny without the deletion and (ii) that a shift to smaller polysomes is detected in the two segregants with the copy 2 deletion. Taken together, these two observations suggest that a depletion of 40S subunits due to the deletion of SJ6A-2 (which codes for a small subunit protein) results in fewer translational initiation events and that this, in turn, has a direct effect on the growth rate. Again, as has been demonstrated with other duplicated yeast RPs (1, 11, 18), this illustrates that the presence of the remaining active gene for the small subunit protein in question, SJ6A-1, does not compensate at any level for the deletion of its duplicate copy, nor does the decrease in 40S subunit assembly appear to signal a concomitant decrease in manufacture of 60S subunits that are actually present in excess in the copy 2-deleted haploids. Are RP28 and S16A gene products essential for cell viability? We assume that the RP28 gene product is essential for cell viability, since removal of just one copy prevents growth. To determine whether the S16A gene product is also essential, we constructed a diploid strain (CiA-lB/lA) that is heterozygous for the deletion of copy 1 (Ura+) and also

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heterozygous for the deletion of copy 2 (Trp+). This strain also contained an RP28 rescue plasmid (YepRP28-2) required to maintain cells that lacked the essential RP28-1. This diploid was sporulated, and a random spore analysis was performed. Haploid progeny were plated on complete medium lacking leucine (to maintain the RP28 rescue plasmid). Single colonies were then tested for mating type to confirm haploidy and replica plated to Ura- and Trpmedium. If the S16A gene product is not essential for growth, then approximately one-fourth of the colonies should be Leu+, Ura+, and Trp+. These haploids would have both copies of S16A missing, with the essential RP28 function being provided by a plasmid-encoded gene. The fact that no Leu+, Ura+, or Trp+ colonies are present of 37 colonies tested indicates that S16A is essential for viability. Finally, in all cases thus far, the act of removing a functional yeast RP from the cell either by gene replacement or by another similar method has resulted in lethality (L3 [4], L29 [4, 12], RP5JA and B [1], L16A and B [18], and L2A and B [11]). This is in contrast to Escherichia coli where at least 16 functional RP genes of 52 can be removed without a lethal outcome (3). A trivial explanation of this difference could be that we simply have not tested enough yeast RP genes. On the other hand, eucaryotic RPs may have a more fundamental role in the function of the ribosome than their procaryotic counterparts and, therefore, cannot be eliminated. In this regard, it has been suggested that RPs may play only a minor role in bacterial ribosome function (partially on the basis of the fact that so many can be omitted) because rRNA performs most of the important catalytic functions (14). The results of deletion studies such as this could indicate a more essential functional role for RPs in eucaryotic ribosomes.

6. 7.

8. 9. 10.

11.

12.

13.

14.

15. We thank Fran Baldwin and Audrey Ellis for their assistance in preparing the manuscript. The contribution of Tom McQuade and Alice Ho in performing some of the tetrad dissections and cell

growth experiments is greatly appreciated. This work was supported in part by Public Health Service grant GM 31812 to N.J.P. from the National Institutes of Health, by an American Cancer Society Institutional Research grant no. 1N174B, and by Designated Research Funds from the Department of Biological Sciences at University of Maryland Baltimore County. LITERATURE CITED 1. Abovich, N., L. Gritz, L. Tung, and M. Rosbash. 1985. Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 5:3429-3435. 2. Botstein, D., S. C. Falco, S. E. Stewart, M. Brennan, S. Scherer, D. T. Stinchcomb, K. Struhl, and R. W. Davis. 1979. Sterile host yeast (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene 8:17-24. 3. Dabbs, E. R. 1986. Mutant studies on the prokaryotic ribosome, p. 733-748. In B. Hardesty and G. Kramer (ed.), Structure, function, and genetics of ribosomes. Springer-Verlag, New York. 4. Fried, H. M., H. G. Nam, S. Loechel, and J. Teem. 1985. Characterization of yeast strains with conditionally expressed variants of ribosomal protein genes tcml and cyh2. Mol. Cell. Biol. 5:99-108. 5. Herruer, M. H., W. H. Mager, L. P. Woudt, R. T. M. Nieuwint,

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