Most of the yeast genomic sequences are not essential for cell growth and division

Cell, Vol. 46, 983-992,

September

26, 1986,

Copyright

0 1986 by Cell Press

Most of the Yeast Genomic Sequences Essential for Cell Growth and Division Mark G. Goebl’ and Thomas D. Petes Department of Molecular Genetics and Cell Biology University of Chicago Chicago, Illinois 60637

Summary To determine the fraction of the yeast Saccharomyces cerevisiae genome that is required for normal cell growth and division, we constructed diploid strains that were heterozygous for random single disruptions. We monitored the effects of approximately 200 independent disruptions by sporulating the diploids and examining the phenotype of the resulting haploid strains. We found that only 12% of the disruptions were haploid-lethal, 14% resulted in slow growth, and an additional 4% were associated with some other new phenotype (such as an auxotrophic requirement). No obvious new phenotype was detected for 70% of the disruptions. Introduction All eukaryotes appear to have more DNA than is necessary to encode essential cellular functions. This statement is based on three types of evidence: physical analysis of genomic DNA, studies of the fraction of the genome that is transcribed, and genetic estimates of the number of essential genes. One argument that eukaryotic cells may contain inessential DNA is that the amount of DNA in a eukaryotic cell (the C value) does not always correlate with the complexity of the organism as measured by other criteria. For example, the African lungfish Amphiuma has about 30 times more DNA per cell than the human, a presumably more complex organism (Pedersen, 1971). This lack of a simple relationship between the amount of DNA per cell and the complexity of the organism has been termed the “C value paradox” (reviewed by Thomas, 1971; Lewin, 1980). Since eukaryotic genomes contain both single-copy and repeated DNA (Britten and Kohne, 1968), one explanation of this paradox is that organisms that have anomalously large C values have proportionally large amounts of repeated DNA. Although for some classes of organisms this explanation appears valid (Gall and Atherton, 1974), for others it does not. For example, the amphibian Necturus maculosis has ten times more single-copy DNA per cell than the human (Baldari and Amaldi, 1976). A second line of evidence indicating that some eukaryotic DNA is not essential derives from studies of transcription. In most higher eukaryotes that have been examined (reviewed in Lewin, 1980), fewer than 20% of the single-copy DNA sequences are transcribed into poly-

Are Not

somal mRNA. It is difficult to estimate from these studies the fraction of essential DNA, since it is unlikely that all genes that are transcribed are essential. In addition, it is clear that some regions that are not transcribed (such a8 promoters) are essential. Despite these uncertainties,.the transcription studies support the notion that a substantial fraction of the eukaryotic genome may be inessential. Similar conclusions were also reached in genetic studies. Lefevre (1974) found that about half of the X-ray-induced breakpoints of chromosome rearrangements in Drosophila melanogaster were not associated with a lethal effect. Although the interpretation of these results is complicated by the nonrandom nature of breakpoints induced by X rays (Lifschytz, 1978), these results suggest that about half of the Drosophila genome may be inessential. In both Caenorhabditis elegans (Brenner, 1974) and D. melanogaster (Judd et al., 1972) genetic studies have shown that there is only one essential gene per 30 kb of DNA. In addition, Gausz et al. (1986) in examining a 315 kb region of the Drosophila chromosome, were able to detect 46 transcripts but only 12 complementation groups. In summary, several lines of evidence indicate that many higher eukaryotes contain a large amount of DNA that has no obvious essential function. We decided to investigate the amount of essential DNA in a lower eukaryote, the yeast Saccharomyces cerevisiae. For several reasons, the yeast genome would be expected to be “lean” in comparison with the genomes of higher organisms. First, the genome is small (about 14,000 kb) and contain8 mostly (90%) single-copy DNA (Petes, 1980). Second, a large fraction of the genome is transcribed (about 50%-60%; Hereford and Rosbash, 1977; Kaback et al., 1979), and relatively few yeast genes contain intervening sequences (Petes, 1980). Thus, one might predict that most of the DNA in the genome would be essential. Prior to our study, Kaback et al. (1984) did an experiment that indicated that yeast might have relatively few essential genes. Using a procedure designed to select temperature-sensitive mutants on one yeast chromosome (chromosome I), they found that 32 independent ts mutants mapped to only three complementation groups. Since it is not clear to what extent most essential genes are mutable to temperature-sensitive alleles, we decided to reinvestigate this issue using a different approach. We took advantage of the observation that transforming DNA integrates into the yeast genome by homologous recombination (Hinnen et al., 1978; Rothstein, 1983) to construct diploid strains that were heterozygous for random single disruptions of the genome. By determining what fraction of these disruptions were haploid-lethal, we estimated the fraction of the genome that was essential. We found that only 12% of the yeast genome is essential under standard laboratory conditions. Results

* Present address: Department Seattle, Washington 98195.

of Genetics,

University

of Washington,

The basic strategy used in these studies was to create a large number of diploid yeast strains, each strain hetero-

Cell 984

Construction of the Diploid Host Yeast Strain MGG3 The disruptions were made in the diploid yeast strain MGG3. As described in Experimental Procedures, this strain was derived from the haploid strain SJR14 (genotype a ura3-52 leu2-3,112 his3-Al) by a mating-type switch. Thus, the diploid MGG3 is homozygous at all genetic loci except mating type. The diploid MGG3 had a number of advantages for subsequent studies. First, the homogeneous genetic background allowed us to detect subtle (10%) effects of disruptions on the growth rate of spores. Second, the excellent viability of spores derived from MGG3 (greater than 95%) facilitated characterization of gene disruptions that resulted in spore inviability. In addition, the strain is homozygous for nonrevertible mutant alleles of three genes (ura3, his9 and leu2) for which the wild-type genetic alleles have been cloned. These three wild-type genes were used to construct the heterozygous disruptions.

Bamtll

HamHI

Cleave

wirh

Figure 1. Construction placement

8~mlll

and

of a Heterozygous

transform

Gene

yeast

Disruption

by Trans-

When a recombinant plasmid containing a BamHl fragment of yeast DNA (into which is inserted a wild-type UFfA3 gene) cloned at the BamHl site of pER322 is cut with the EarnHI enzyme, two linear fragments are produced. When these linear fragments are used to transform a diploid yeast strain, the yeast DNA fragment containing the URA3 gene recombines with homologous sequences on one of the two homologous chromosomes of the diploid (Rothstein, 1983). The result of this recombination event is a diploid strain that is heterozygous for a single disruption.

zygous for a single disruption. The disruptions were constructed by inserting a selectable yeast gene into the genome by transformation. The diploid cells were allowed to undergo meiosis. If the individual diploid yielded four spores that gave rise to four colonies of normal size, we concluded that the disruption interrupted an inessential region of the yeast genome. If the diploid gave rise to four spores yielding two colonies of normal size and two small colonies (containing the disrupting sequences), we concluded that the disruption interrupted a region of the genome that was required for normal growth but was not essential for viability. If the diploid gave rise to only two colonies per tetrad, and the cells in these colonies lacked the selectable gene used to make the disruption, we concluded that the disruption interrupted an essential region of the genome. By looking at a large number of such diploids, we could calculate the fraction of the yeast genome that is essential for viability. We discuss our analysis in three parts. First, we describe the construction of the diploid that was used as a host for the disruptions. Second, we describe experiments in which the disruptions were made using restriction sites. Third, we discuss results obtained when disruptions were made using transposable elements.

Construction of Heterozygous Disruptions in the Diploid Strain MGG3 Using Restriction Sites The construction of strains (derived from MGG3) containing different single genomic disruptions was done in several steps. First, we constructed recombinant plasmids containing BarnHI fragments of yeast DNA from the strain SJR14 inserted into the BamHl site of pBR322; since the tandemly repeated yeast ribosomal RNA genes lack BamHl restriction sites, our use of this enzyme avoided repeated construction of plasmids containing this family of genes. Second, we analyzed 57 of these plasmids individually for those yeast DNA insertions that had a single restriction site for one of the following restriction enzymes: 89111, Bell, Xbal, or Xhol. The plasmids were examined for these particular sites since these sites are compatible with restriction fragments containing the selectable genes URA3 (Bglll, Bell, and Xbal) and LEUP (Xhol), and since these sites do not appear in either pBR322 or in the selectable genes used in creating the disruptions. Of the 57 plasmids examined, 29 had single sites for one of the four restriction enzymes. The third step in the construction was the insertion of either URA3 or LEU2 into each of the 29 plasmids. Each of these plasmids was then treated with BamHl and, in individual transformation reactions, introduced into the diploid MGG3. The treatment of the recombinant plasmid with BamHl splits the plasmid into two fragments, a fragment containing only pBR322 sequences and a random BamHl fragment of the yeast genome into which an insertion of either LEU2 or UR43 has been made. It has been shown by Rothstein (1983) that when yeast cells are transformed with a linear DNA fragment containing an insertion, this fragment integrates by homologous recombination into the genome and displaces the resident undisrupted chromosomal segment (a “transplacemerit”; Figure 1). For each of the 29 plasmids constructed, we obtained a yeast transformant derived from MGG3. Transformants were obtained with normal efficiencies for all 29 plasmids, indicating that none of the disruptions had a dominant lethal phenotype. The mitotic stability of the transformed

Essential 985

Yeast

Genes

Figure 2. Analysis of Colonies Transformed Diploid Strains

Derived

from Tetrads

of Three

Different

In the photographs, each numbered row represents colonies derived from spores of a single tetrad. Following tetrad dissection, each plate was incubated for 3 days at 32% before being photographed. (A) Seven tetrads derived from the transformed diploid MGG3-E6::URA3. This disruption had no obvious effect on the growth rate. In each tetrad, two of the spores contained the disruption and two did not. (B) Seven tetrads derived from the diploid MGG3-F12::URA3. All of the complete tetrads on this plate had two spores that gave rise to colonies and two that did not. The live spores were Ura-, indicating that the USA3 insertion is within an essential gene. (C) Seven tetrads derived from the diploid MGG3A12::URA3. All complete tetrads on this plate segregated two slow-growing strains to two strains that grew at normal rates. All slow-growing strains were Ura+. and all normal-growing strains were Ura-.

phenotype was checked by streaking the transformants on rich growth medium and then replica-plating the resulting colonies to media lacking either leucine or uracil. In addition, DNA was isolated from the transformed diploid strains and examined by Southern analysis using pBR322 as a hybridization probe. Any transformed strains that had a mitotically unstable Ura+ or Leu+ phenotype or that hybridized to pBR322 were not examined further, since these properties are those expected if the transforming

DNA had not integrated into the host genome by transplacement. For each of the 29 transformed diploid strains, at least seven tetrads were dissected. In examining colonies derived from the dissected spores after two days on rich growth medium, three different classes of transformant were observed (Figure 2). In class I transformants, the four spores of a single tetrad gave rise to colonies of approximately the same size. In class II transformants, two of the spores gave rise to colonies of normal size and two of the spores gave rise to smaller colonies. In class Ill transformants, only two of the four spores gave rise to detectable colonies. To ensure that these phenotypic effects were a result of the disruption, rather than a secondary effect of the transformation, all colonies were also replica-plated to media lacking leucine or uracil. For most of the transformants, the phenotype (either the lethality or the slow-growth effect) cosegregated with the Ura+ or Leu+ phenotype in all tetrads. For some of the transformants, however, the transformed strains contained a lethal mutation that did not cosegregate with the disruption. For such transformants, we either analyzed a second transformant constructed using the same plasmid or reconstructed a heterozygous diploid by crossing a Ura+ or Leu+ spore from the first transformant with an isogenic untransformed haploid. Of the 29 transformants, 22 were class I transformants, three were class II transformants, and four were class Ill transformants (Table 1). We also tested at least two complete tetrads derived from 25 of the transformed diploids (the disruptions that were haploid-lethal could not be tested) for the following properties: sensitivity to ultraviolet light; sensitivity to the chemical mutagen methyl methanesulfonate (MMS); ability to mate; osmotic sensitivity; ability to grow on minimal growth media; ability to grow anaerobically; ability to grow at 42% and 16%; ability to respire; and ability of diploids homozygous for the disruption to sporulate. These tests detected very few new phenotypes (Table 1). The haploid spores derived from the transformants MGG3-AS::URA3 and MGG3-Gll::URA3 were not respiratory-competent. Thus, in these strains, the slow growth is likely to be the result of a mutation affecting mitochondrial function (a pet gene) (Dujon, 1981). The only other new phenotype found in these experiments was that haploid strains containing the disruption derived from MGG3G5::URA3 had a requirement for phenylalanine. We showed by complementation tests that this strain was mutant in the pha2 gene, a gene encoding prephenate dehydratase (Lingens et al., 1966). In conclusion, when 29 independent disruptions were examined, four disruptions were haploid-lethal, three resulted in slow growth (two of which were pe+ mutants), and one resulted in a new auxotrophic requirement. One possible explanation of the lack of effect of most of the disruptions is that the disruptions had occurred within repeated genes. This possibility was checked by performing a Southern analysis of DNA isolated from one tetrad of each of the 25 transformed diploid strains in which the disruption was not haploid-lethal; for those four

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Table

Name

1. Genetic

and Physical

of Transformant

Analysis

of Yeast

Size of BamHl Fragment (kb) before Disruption

Strains Restriction Used lo Disruption

Containing Site

Create

Gene

Disruptions

Phenotypic

Class of Disruptiona

Constructed

Insertion in Single-Copy or Repeated DNAb

3.0 2.9 1.1 8.2 5.5 2.8

Bell Bell Bglll Bglll Bglll Bglll

I I I I I II

SC

5.2 1.4 5.0 3.2 6.1 2.6 3.5 3.1 8.4 5.0 5.0 10.0 6.1 2.5 2.6 1.2

Bglll Xhol Bglll Xhol Xbal Xhol Bglll Xhol Xhol Xhol Bglll Xhol Xbal Xhol Bglll Bglll

II I I I I I I I I I I I I I I I

SC(R) SC SC SC SC SC SC SC

WC) WC) SC SC SC R

8.6 3.1

Bglll Bell

Ill I

SC SC

MGGJ-G8::URAB MGG&GlO::URAS MGGS-Gl 1 ::URAS

12.0 9.1 2.8

Xbal Xbal Bglll

I III II

SC(R) SC SC

MGG3-H4::LEUZ MGGS-HG::URA3

12.5 2.1

Xhol Bglll

Ill Ill

SC SC

MGG%A2::URA3 MGG3A3::URA3 MGG3-A4::fJRA3 MGG3-AG::URA3 MGG3-A7::URA3 MGGS-A9::URAS MGG3-AI2::URA3 MGG3-Bl::LEU2 MGG3-B3::lJRA3 MGG3-B5::LEU2 MGGJ-ClO::LJRAS MGGS-Cl I ::LEUP MGGS-D12::LJRA3 MGG3-El::LEU2 MGG3-E3::LEU2 MGG3-E4::LEU2 MGG3-EG::URA3 MGG3-E8::LEU2 MGGS-E9::URAS MGG3-Ell::LEUZ MGGB-FS::URA3 MGGI-FlO::URAS MGGJ-Fl2::URA3 MGG3-G5::URA3

Restriction

Other

Enzyme

Sites

Comments

SC(R) SC SC(R) SC SC

Petite; same disruption MGG3 Gl 1 ::URAS

as

SC(R) SC

a Class I disruptions have no effect on colony size, class II disruptions result text for details. b SC indicates that the BamHl fragment used to make the disruption contains single-copy and repeated DNA, and the insertion is within the single-copy DNA. ed DNA, and the insertion is within the repeated DNA. R indicates that the

strains in which the disruption was haploid-lethal, we examined DNA isolated from the diploid. For this analysis, the genomic DNA was treated with the restriction enzyme BamHI, and the hybridization probe was the undisrupted BamHl fragment (contained within pBR322) used in the construction of that specific transformant. Three patterns of hybridization were expected (and observed) in the analysis of the different transformants. If the BamHl fragment used in constructing the disruption contains only single-copy DNA, then the strains without the disruption should have a single band of hybridization that is identical in size to the original BamHl fragment. The strains containing the disruption should lack this band and have a new band representing a larger BamHl fragment. This pattern of hybridization was observed for 21 of the 29 transformants (Table l), and one example of a Southern analysis of this type of transformant is given in Figure 3. The second observed pattern of hybridization was that the BamHl fragment used in making the disruption hybridized to multiple BamHl fragments in genomic DNA iso-

Using

in small colonies,

Insertion Insertion

in Y’ repeat in Ty element

Insertion repeat

in uncharacterized

Requires phenylalanine; pha2 mutation

Petite; same disruption MGGS-A9::URAS

and class

III disruptions

as

are haploid-lethal.

See

only single-copy DNA. SC(R) indicates that the fragment has both R(SC) indicates that the fragment has both single-copy and repeatentire BamHl fragment is repeated.

lated from all spore cultures. This is the pattern expected if the BamHl fragment used in constructing the disruption includes both single-copy and repeated DNA; seven of the 29 transformants had this pattern of hybridization (Table 1). To determine whether the insertion was within the repeated portion of these BamHl fragments, we repeated the Southern analysis for these seven transformants and used hybridization probes derived from each side of the point of insertion of the selectable genes into the BamHl fragment. If the disruption is within single-copy DNA, only one of these two fragments should hybridize to repeated DNA. By this criterion, in five of the seven transformants, the selectable gene was inserted within single-copy DNA. In two of the transformants (MGGS-EG::URA3 and MGGSE8::LEU2), the insertion was within the repeated portion of the insert. Detailed restriction maps of these BamHl fragments (data not shown) indicated that the insertion in MGG3-EG::UFtA3 was within the telomeric Y’ repeat (Chan and Tye, 1983), and the insertion in MGG3-E8::LEU2 was within a Ty element (Roeder and Fink, 1983). The third pattern of hybridization was observed for only

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Yeast

Genes

ABCDE

Figure 3. Southern Containing a Gene

Analysis Disruption

of a Transformant

(MGG3-Bl::LEU2)

Into a recombinant plasmid containing a 1.35 kb BamHl fragment of yeast DNA (pMG-Bl), we inserted a 2.2 kb Sall-Xhol fragment containing the yeast LEU2 gene. The insertion was made in a Sal1 site within the BamHl fragment. This plasmid was treated with BamHI, and the resulting linear fragments were used to transform the diploid yeast strain MGGS. The resulting diploid (MGGS-Bl::LEUP) was sporulated, and tetrads were dissected. DNA was isolated from the original transformed diploid (lane A) and from four haploid strains derived from a single tetrad (lanes B-D). The DNA was treated with BamHl and was examined by Southern analysis, using pMG-Bl as a hybridization probe. As expected, the diploid showed two bands of hybridization, one representing the undisrupted 1.35 kb BamHl fragment and one representing the 3.55 kb disrupted BamHl fragment. Each haploid strain had only a single band, and the 3.55 kb band cosegregated with the Leu+ phenotype.

one of the transformed strains (MGG3-FlO::URAS). In Southern analysis of BamHl fragments derived from strains without an insertion, a single BarnHI fragment of the expected size was observed. In analysis of BamHl digests of haploid strains containing an insertion, two bands of hybridization were detected, one of the size expected for the undisrupted BamHl fragment and one of the size expected for the disrupted fragment; the band representing the undisrupted fragment was about 2-fold more intense than that representing the disrupted fragment. The simplest explanation of this result is that the entire BamHl fragment used to construct the disruption in MGG3-FlO::URA3 is repeated three times per haploid genome and that we have disrupted a single one of these repeats. By using restriction enzymes other than BamHl (data not shown), we confirmed that the sequences derived from MGG3-FlO::Uf?A3 were repeated even in untransformed strains. The Southern analyses that were done to characterize the transformed strains (Table 1) also rule out the possibility that the transformation procedure often results in a duplication of DNA sequences rather than a replacement.

If, in addition to the BamHl fragment containing the disruption, the BamHl fragment containing the undisrupted DNA sequences were present in a haploid cell, bands of hybridization representing each of these fragments would be detected by Southern analysis. When we examined the DNA isolated from haploid transformants (that did not hybridize to pBR322 DNA), this pattern was observed only for one transformant, MGG3-FlO::URA3. We conclude, therefore, that stable transformants that fail to hybridize to pBR322 DNA usually represent true transplacements, confirming the results of Rothstein (1983). There are two problems in our estimates of the fraction of essential yeast DNA. One problem is that the number of disruptions studied was small. A second problem is that it is not clear that restriction sites for the enzymes used to create the disruptions are distributed randomly with respect to essential and inessential regions of the yeast genome. Although studies done in other organisms indicate that restriction sites appear to be randomly placed in single-copy sequences (Hamer and Thomas, 1975), no such study has been done in yeast. In addition, two of the enzymes used in our study (Bell and Xbal) have nonsense codons in the recognition sequence and, therefore, in at least one reading frame, will avoid coding regions. Although these considerations are unlikely to affect our conclusions substantially, we decided to use a different procedure to create disruptions. Construction of Heterozygous Disruptions in the Diploid Strain MGG3 Using Transposon Mutagenesis Seifert et al. (1988) and Snyder et al. (1988) have described techniques for generating disruptions of cloned yeast DNA sequences in vivo in Escherichia coli using transposon mutagenesis. In the system described by Seifert et al., a Tn3-derived transposon that contains a selectable yeast gene is used to make insertions in yeast DNA sequences cloned in the plasmid vector pHSS8. In our experiments, we inserted BamHl fragments of yeast DNA isolated from MGG3 into the BamHl site of pHSS8, and we used the derivative of Tn3 that contained the wildtype yeast HIS3 gene (mini-Tn3(H/S3)); other details are given in Experimental Procedures. We isolated DNA from 20 individual plasmids containing insertions of yeast DNA (with the Tn3-generated disruption) for yeast transformation experiments. We did a detailed restriction analysis of 18 of these 20 plasmids using BamHI, Mspl, and Smal and Xbal to determine whether the plasmids represented a random sampling of the yeast genome. We found that the average size of the BamHl fragments in these plasmids (3 kb) was somewhat smaller than that observed in the study in which disruptions were made using restriction enzyme sites (6 kb), suggesting that one or more steps in the disruption procedure selects in favor of plasmids with small insertions. Restriction analysis, however, indicated that all but two of the plasmids contained different yeast DNA insertions. Thus, the plasmids do not represent a highly selected subset of the yeast genome. The 20 plasmids described above were treated with the restriction enzymes Smal and Xbal and, in separate ex-

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Table

2. Summary

of Effects

of Random

Gene

Experiment

Total

Disruptions made with restriction sites Individual disruotions made with Tn3 Disruptions made en masse with Tn3 Total

29 20 167 216

Disruptions Disruptions (100%) (100%) (lOO%j (100%)

in Yeast Haploid 4 4 18 26

periments, were transformed into the yeast diploid strain MGG3; His+ transformants were selected. The enzymes Smal and Xbal were chosen because they cut pHSS6 in the polylinker that flanks the BamHl site into which the yeast DNA is inserted and do not cut the miniTn3(his3) transposon (Seifert et al., 1986). Thus, these enzymes should generate linear DNA fragments containing either yeast DNA with the transposable element (and a small amount of polylinker) or pHSS6 DNA. The linear fragment of yeast DNA containing the transposon should be capable of recombining with chromosomal sequences to produce the heterozygous disruption in the diploid (Rothstein, 1983). The small amount of the polylinker, which represents sequences that have no homology to yeast DNA, does not prevent this recombination event, although the efficiency of the recombination is somewhat reduced (Seifert et al., 1986). We found that all 20 plasmids containing disruptions produced by the transposon were capable of transforming MGG3 to the His+ phenotype. The resulting transformed strains were analyzed in the same way as described previously. When the 20 strains were sporulated and dissected, we found that there were 13 class I transformants, three class II transformants and four class Ill transformants (Table 2). We examined all haploid strains for the other previously described mutant phenotypes, except for osmotic sensitivity and ability of the homozygous diploid to sporulate. The only new phenotype detected in these tests was that one of the disruptions that resulted in slow growth also made the cells sensitive to the mutagen MMS. It is clear that this method of generating disruptions yields results similar to those obtained using restriction sites. Since these experiments suggested that the transposon disruption procedure was working properly, we transformed the yeast strain MGG3 en masse with yeast DNA fragments containing insertions of the mini-Tn3(H/SS) transposon. For this experiment, we isolated DNA from a mixture of the 20,000 E. coli strains that contained plasmids with BamHl fragments of yeast DNA into which transposons had been inserted. Approximately 200 His+ yeast transformants were purified. Before analysis of spores derived from the diploid transformants, we did a preliminary characterization of the diploid strains. We first examined the mitotic stability of the His+ phenotype. Eleven of the transformants were mitotically unstable and were not characterized further. In addition, DNA was isolated from each diploid transformant and was hybridized to the plasmid vector pHSS6; 34 of the transformants hybridized to pHSS6 and were not analyzed further. The remaining MGG3 diploid transformants were sporulated, and three tetrads from each transformant were dissected. As expected from previous experiments, most

(14%) (20%) (11%) (12%)

Lethal

Slow Growth

New Phenotype

No Effect

3 3 25 31

1 0 7 8

21 13 117 151

(10%) (15%) (15%) (14%)

(3%) (0%) (40/o) (4%)

(72%) (65%) (70%) (70%)

(90%) of the diploid strains analyzed could be classified as class I, II, or Ill transformants. In addition to these classes, two other types of transformant were found at low frequency. We found ten strains in which more than one tetrad segregated either 3+:1- or 4+:0- for the histidine requirement. Such strains represent double disruptions, presumably arising as a result of a yeast cell taking up two fragments of DNA during transformation. In tetrads derived from double transformants, if none of the spores showed a new phenotype, we counted each strain as representing two class I transformants. If two of the spores were slow-growing in the double transformant, we mated the slow-growing His+ spores to an untransformed haploid of the opposite mating type. If the His+ phenotype cosegregated with the phenotype of slow growth in this backcross, we counted the double transformant as representing one class I transformant and one class II transformant. The last, unexpected class of diploid transformant (seven strains) included those in which only two of the four spores were viable but the loss of viability did not segregate with the His+ insertion. Such strains could represent either a double insertion of the transposon into the yeast genome (one in an essential region and one in an inessential region), or a single insertion of the transposon into an inessential region of the genome in a strain containing a recessive lethal mutation that was not generated by insertion of a transposon. These two possibilities can be distinguished by Southern analysis of genomic DNA isolated from the diploid strain and the derived haploid spores (data not shown). We found that three of these strains contained a single insertion that was not associated with the lethal phenotype, and four of the strains contained two insertions, one associated with the recessive lethal phenotype and one unassociated. Although the appearance of a recessive lethal phenotype that is unassociated with a disruption in three strains may appear surprising, other workers (Shortle et al., 1985) have also found that transformed yeast cells have a higher-than-normal level of spontaneous mutations. As in the previous experiments, we examined strains containing haploid-viable disruptions for other phenotypes. We did the same analyses described previously except we did not test the strains for osmotic sensitivity, ability to grow anaerobically, or ability of the homozygous diploid to sporulate. Two of the 25 strains that grew slowly lacked mitochondrial function. We found seven of the Class I strains had a new mutant phenotype induced by the disruption: four had whiter colonies than the wild type, two of the disruptions resulted in cells that formed larger colonies than the wild type, and the remaining disruption mutant was more sensitive to MMS than the wild type. In

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Genes

summary, of the 167 individual disruptions examined in the experiment with the pooled preparation of plasmids, 18 resulted in haploid lethality, 25 resulted in slow growth, seven generated a new phenotype other than lethality or slow growth, and 117 had no obvious effect (Table 2). We did a limited physical analysis of about 50 randomly chosen class I and class II individual transformants obtained in the en masse experiment. DNA from 50 different haploid strains containing the Tn3-induced disruptions was treated with EcoRl and then examined by Southern analysis using the HIS3 gene (cloned in pBR322) as a hybridization probe. As expected, most (47 of 50) of these strains contained more than one band that hybridized to HIS3. DNA from all strains hybridized to a band of approximately 10 kb, representing the HIS3 gene at its normal location on chromosome XV (Struhl and Davis, 1980). In addition, most of the strains contained two additional bands of hybridization representing the insertion of miniTrG(HIS3) into other positions in the genome. Two extra bands of hybridization were expected because there is an EcoRl site within the mini-Tn3(H/S3) (Seifert et al., 1988). The position of these bands was different in different transformants. No obvious duplications were observed in these hybridization patterns, suggesting that there is no large bias in favor of recovering certain classes of transformant. These results also indicate that the most of the His+ strains obtained in these studies must represent transformation events rather than reversion or gene conversion events at the his3 locus. Discussion The data from the study are summarized in Table 2. We conclude that 12% (8%-170/o, within 95% confidence limits; Pearson and Hartley, 1986) of the disruptions are haploid-lethal, 14% (lo%-20%) result in slow growth, and 4% (2%-7%) result in some other new phenotype. We find that 70% of the disruptions have no obvious effect. Since this conclusion is somewhat surprising, we must consider alternative explanations for our results. One possibility is that the disruptions are not random, insertions being made preferentially in regions of the genome that are not essential. We believe that this possibility is unlikely, since two independent methods of generating insertions gave similar results (Table 2). In addition, the Tn3 element has relatively little site specificity; in experiments done with the same derivative of Tn3 used in our studies, all 32 independent insertions into a cloned fragment of 2 kb were different (Seifert et al., 1986). A second possibility is that the insertions used to create disruptions do not inactivate the gene into which they are inserted. Since the insertions contain both transcriptional and translational termination signals, we believe that most insertions will inactivate, although some of the insertions near the 3’ end of the gene may allow partial activity. This assumption is supported by the observation that the coding region of the adel gene determined using insertions of mini-Tn3 (HIS3) is the same as that defined by R-looping studies (Seifert et al., 1986). A third possible explanation for our data is that most yeast DNA sequences in the haploid yeast genome are

represented more than once. This possibility is unlikely, because the detailed Southern analysis that we did of 29 transformants indicated that most of the disruptions were within single-copy sequences. We cannot rule out the possibility that many yeast sequences are duplicated but have sufficient sequence divergence between the repeats that they do not cross-hybridize under the conditions used in our study. None of the effects described above appears likely to represent an important source of error in our studies. There ae several more likely explanations of our observations. First, there may be genes that encode functions that have a subtle but significant effect on growth rate. We found that 10% differences in growth rate resulted in colonies of different sizes in our experiments; smaller growth rate differences might be harder to detect. Second, it is possible that many yeast genes encode functions that are duplicated by structurally unrelated genes. Thrash et al. (1985) have suggested that the yeast cells containing a mutation in the topoisomerase I gene are viable because the activity of topoisomerase II can compensate for the defect. Third, there may be genes encoding functions that are important in the wild but that were not measured by any of the tests we performed. Fourth, much of the yeast genome may consist of DNA sequences that are structurally important but do not encode gene products. Disruption of such sequences may not lead to an obvious effect. One argument against this possibility is that one would expect DNA sequences that are structurally important to be repeated genes. In addition, since over 50% of the yeast genome is transcribed (Kaback et al., 1979) it is unlikely that two-thirds of the genome consists of structural DNA. Although it is likely that all of the above possibilities contribute, in part, to the lack of effect observed for many of the genomic disruptions, we believe that a fifth possibility also exists: a substantial fraction of the yeast genome may have no useful function. This interpretation of our results raises other questions. How did nonfunctional DNA sequences become established in the yeast genome? Since yeast, like all organisms, has an evolutionary history, one explanation of the existence of nonfunctional DNA is that these sequences were derived from species in which the DNA was functional, but these functions are no longer relevant to the present species; by this interpretation, these sequences are analogous to vestigial organs. Although one might expect natural selection to favor yeast cells that have lost the functionless DNA, deletion events in yeast are very infrequent. In addition, it is not clear to what degree selection acts against cells having excess DNA. Although several interpretations of our results are possible, we favor the possibility that much of the yeast genome is not functionally important. It should be stressed that our study concerns the fraction of the genome that is essential, and it does not directly address the issue of the number of essential genes. Making the following assumptions, however, we can estimate the proportion of essential yeast genes: first, all transcribed regions of the genome represent genes; second, the average yeast transcript is about 1.3 kb in size (J. Pringle, personal communication); third, the genome is

Cell 990

50%-60% transcribed (Kaback et al., 1979; J. Pringle, personal communication); fourth, the yeast genome (excluding ribosomal DNA) is 13,000 kb (reviewed by Petes, 1960); and fifth, all disruptions that result in a new phenotype are within transcribed regions. Using these figures, we calculate that yeast has about 5500 genes: 1200 essential genes, 1400 genes required for optimal growth rates (presumably essential in the wild), and 400 genes affecting other phenotypes. Kaback et al. (1964) showed that there were only a small number of genes on yeast chromosome I that were mutable to temperature-sensitive lethality. One interpretation of their results (based on estimations of the proportion of genomic DNA on chromosome I) was that there are only 250 essential genes in the yeast genome. Based on our data, it is more likely that the apparently small number of essential genes may reflect “hot spots” for the mutagens used, the inability to mutate essential genes to temperature sensitivity, structural duplication of essential genes, or an unusually small number of essential genes on chromosome I (as suggested by Kaback et al., 1964). Since the yeast genome is small and contains little repeated DNA, one might expect that similar experiments done in other eukaryotes with larger genomes would detect an even smaller fraction of essential sequences. In studies done in Drosophila, estimates of the fraction of essential DNA differ widely. Although Lefevre (1974) found that approximately half of the X-ray-induced translocation breakpoints were associated with a lethal effect, A. Spradling (personal communication) found that only eight of 55 insertions of a P element into the Drosophila third chromosome were lethal when homozygous. G. Rubin (personal communication) has found that approximately one-third of P element insertions were homozygous-lethal, but Simmons et al. (1965) observed that only 1% of P element insertions caused a recessive lethal mutation. The reasons for these differing values are not clear. The estimate of essential sequences based on translocation data may be too high because of position effects inactivating genes near the breakpoint of the translocation. The P element experiments may be affected by the specific element used, the strain of Drosophila, or the presence of extraneous lethal mutations linked to the P element insertion (as suggested by Simmons et al., 1985). Although the data are more limited for mammals, they indicate that a smaller fraction of the genome is essential than that observed in yeast. Ft. Jaenisch (personal communication) found that insertional mutagenesis with retroviruses in mouse embryos at the preimplantation stage resulted in one recessive lethal line in 40 examined. In other experiments, in a collection of seven mouse lines obtained by injecting DNA sequences into fertilized eggs, one nonlethal mutation was observed; six lines had no obvious mutations (Woychik et al., 1985). In conclusion, a number of different studies indicate that much of the eukaryotic genome has no obvious function. Inessential DNA is found even in organisms, such as yeast and Drosophila, that contain mostly single-copy transcribed genes. Although our studies do not prove that most of the yeast genome has no function (since we can-

not rule out subtle growth effects or unanticipated functions), our research clearly demonstrates that only a small fraction of the yeast genome is being examined in studies of traditional types of mutations. In our study, for example, we detected only one auxotrophic mutation in a sample of 216 disruptions.

Experimental

Procedures

Yeast and Bacterial

Strains

The diploid yeast strain MGG3 used in most experiments was derived from the haploid strain SJR14 (Jinks-Robertson and Petes, 1985), which has the genotype a ho ura3-52 /eu2-3,772 his3-A7 canl. This strain was converted lo a diploid by a mating-type switch using a plasmid (YEpHO) that contains the yeast HO gene (provided by I. Herskowitz, University of California, San Francisco). For testing the allelism of a phenylalanine auxotroph obtained in our studies, we used the yeast strains A334-498 (a /eu2-3 pha2 petx prtl arg8) and A34427B (a /eu2-3 pha2 petx arg8 his ura) (Gaber et al., 1983). The E. coli strains used in the study were HB101 (Maniatis et al., 1982) and GM181 (r~-m~ dam-). All other bacterial strains used in transposon mutagenesis were those recommended by Seifert et al. (1986). DNA Isolation

and Recombinant

Plasmid

Constructions

Yeast DNA was isolated using two different procedures. When large (greater than 10 pg) amounts of DNA were required, we isolated DNA using CsCl gradients containing the fluorescent dye Hoechst 33258 (Petes et al., 1978). For smaller amounts of yeast DNA, the “mini-prep” procedure described by Sherman et al. (1982) was used. Plasmids were isolated using either CsCl and ethidium bromide gradients or cleared lysates (Davis et al., 1980). Several typesof recombinant plasmid were used in our studies. One collection of plasmids was made by inserting random BamHl fragments of yeast DNA (isolated from strain SJR14) into the BamHl site of pBR322 (Maniatis et al., 1982). In the first experiments, these plasmids were screened for those that contained yeast DNA inserts with a single restriction site for Bglll, Xbal, Xhol, or Bell. Using standard methods, we inserted into individual plasmids a 1.2 kb BamHl fragment containing the yeast URA3 gene (into Bglll or Bell sites), a 1.2 kb Xbal fragment containing the yeast URA3 gene (into Xbal sites). or a 2.2 kb Sall-Xhol fragment containing the yeast LEUP gene (into Sall sites). The URA3 gene with BamHl linkers was derived from the plasmid pSR9 (provided by Sue Jinks-Robertson, University of Chicago), and the same gene with Xbal linkers was derived from the plasmid pRA545 (Tatchell et al., 1984). The LEU2 gene was isolated from the plasmid CV9, which was obtained from J. Hicks (Scripps Institute) and is described by Klein and Petes (1984). A total of 29 plasmids were constructed by these techniques (Table 1). In the course of the construction and analysis of the plasmids described above, it became clear that the BamHl fragments used to create the disruptions in the diploid strains MGG3-AS::Uf?A3 and MGG3GIl::URA3 were the same: the other 27 fragments used to construct the disruptions were different from this fragment and different from each other. We calculated the probability of observing two identical BamHl fragments in a collection of 29 randomly chosen fragments by using the following equation: p = 1 - eektk - ‘)‘*N, where k equals the number of plasmids examined (29), and N equals the total number of BamHl fragments in the yeast genome that contain a single restriction site for Bell, Bglll, Xbal, or Xhol. We estimated N to be about 900, based on the frequency with which BamHl is expected to cut yeast DNA and on the fraction of BamHl fragments in our study (29 of 57) that have a single cleavage site for one or more of the four restriction enzymes listed above. Using these estimates, we calculate that there is a 36% chance of obtaining a single duplication in a collection of 29 plasmids. We conclude, therefore, that the BamHl fragments used in this study are likely lo represent a random sampling of the yeast genome. A second procedure (Seifert et al., 1986), employing transposable elements, was also used to make disruptions of cloned yeast sequences.

Essential 991

Yeast

Genes

The yeast BarnHI fragments were cloned in the plasmid pHSS6 and then transformed into an E. coli strain containing a plasmid (pLB101) that encoded Tn3 transposase but did not have a transposable Tn3 element; approximately 10% of the plasmids in our experiments had an insertion of yeast DNA. About 25,000 of these transformants were mated en masse with an E. coli strain that contained a plasmid (pOX38::mTn3) that had a modified Tn3 element. The Tn3 element used in our studies (miniTn3(H/S3)) contained the ends of Tn3, a 8-lactamase gene, the yeast HIS3 gene, and a site (loxp) recognized by the site-specific recombination enzyme encoded by the cre gene. When the plasmid containing miniTnS(HIS3) was introduced into the same cell as the pHSS.6 plasmids (containing yeast DNA) and pLB101, a cointegrate structure between the plasmids containing yeast DNA and pOX38::m-Tn3 was formed. When cells containing these cointegrate structures were mated to an E. coli strain that produced the cre protein, the structures were resolved. After resolution of the cointegrate structures, we transformed the resulting plasmids into HBIOI, selecting about 20,000 bacterial transformants. Plasmid DNA was isolated from approximately 400 individual colonies, treated with the BamHl restriction enzyme, and examined by gel electrophoresis. We found that 55 of the 400 plasmid samples (16%) contained pHSS6 with an insertion of yeast DNA containing the miniTn3 (HIS3). Most of the other plasmid samples contained pHSS6 without yeast DNA but with an insertion of the transposon. Growth Sporulation, and Transformation of Yeast Strains The rich growth medium used in most of our analysis was YPD (Sherman et al., 1982). Both diploid cells and haploid segregants were usually grown at 32OC. The diploid strain MGG3 (and transformed derivatives of MGGI) was sporulated on plates (Sherman et al., 1982) at 23OC. After 3 days, tetrads were dissected by standard techniques, The size of colonies derived from the spores was monitored after 36 hr of growth. Transformation of MGG3 was done according to the procedure of Hinnen et al. (1978). In experiments involving plasmids constructed using restriction sites to create the disruptions, we treated the plasmid DNA with BamHl before transformation. Approximately 5 ug of DNA was used in each experiment. In experiments involving plasmids constructed using transposons, the plasmid DNA was treated with both Xbal and Smal before transformation. Physical and Genetic Analyses of Yeast Transformants To confirm that the transformation procedure created the disruptions as expected, we did a Southern analysis of many of the haploid transformed strains. DNA was isolated (Sherman et al., 1982) and was treated with restriction enzymes (usually BamHI), and the resulting fragments were separated on agarose gels (0.5%) containing ethidium bromide. The separated fragments were transferred to nitrocellulose (Southern, 1975) and were hybridized to a 32P-labeled DNA probe (prepared by nick translation). In analysis of transformants obtained by insertions of LEU2 or URA3 into restriction sites within individual cloned fragments, the probe used in the analysis of the transformants was the plasmid containing the undisrupted BamHl fragment. Thus, each of these yeast transformants was analyzed with a different plasmid probe. In hybridization analysis of transformants derived from the en masse transformation experiment, we used the plasmid pSR7 (Jinks-Robertson and Petes, 1985) which contains the wild-type yeast HI.93 gene inserted into pBR322. Hybridization conditions were standard (Maniatis et al., 1982). In addition to the physical analysis described above, we tested haploid strains containing the disruptions for a variety of other phenotypes: the strains were tested for new auxotrophic requirements by examining their ability to grow on SD minimal medium (Sherman et al., 1982) supplemented only with leucine, histidine, and uracil; mitochondrial function was tested by replica-plating the strains to medium containing the unfermentable carbon source glycerol (Sherman et al., 1982); ability to mate was tested by using laboratory strains (of both mating types) that had complementing auxotrophic mutations; temperature sensitivity and cold sensitivity were tested by replica-plating colonies to YPD plates and incubating at either 42OC or 16°C for about 3 days; sensitivity to ultraviolet light was tested by replica-plating colonies to YPD elates. treating the replica-plated cells with 150 J/m2 of

at 32OCin darkness (Haynes and Kunz, 1961); and sensitivity to MMS was checked by replica-plating cells to YPD containing 0.035% MMS (Prakash and Prakash, 1977). The tests described above were performed with all haploid-viable disruptions. In all of these tests, haploid strains of the same genotype but without the disruption were used as controls. For the 29 strains containing disruptions made at restriction sites, three more tests were done. We tested for osmotic sensitivity by replica-plating cells to YPD medium containing 2.5 M glycerol (Singh and Sherman, 1978). The ability to grow anaerobically was tested by replica-plating cells to YPD plates and incubating the plates overnight at 32OC in a GasPak Disposable Anaerobic System (BSL). To test the ability of diploid strains containing the homozygous disruption to sporulate, we mated individual haploid strains that were of different mating types but contained the same disruption. The resulting diploid strains were incubated on solid sporulation media (Sherman et al., 1982) for 3 days. Acknowledgments We thank J. Pringle, A. Spradling, G. Rubin, P Leder, and Ft. Jaenisch for providing unpublished information. J. Pringle, R. Horvitz, W. Fangman, D. Burke, and all members of the Petes lab are thanked for their commentson the manuscript. We thank Dorothy Stamenovich for technical assistance, F. Heffron and H. Seifert for providing us with the methodology for mini-Tn3 mutagenesis, and T Nagylaki for his advice concerning the statistical analysis of the data. The research was supported by National Institutes of Health research grants (GM24110 and GM34646) and a National Research Service Award (CAO9267-0552) to M. G. G. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘adverfisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

May 27, 1986; revised

July 14, 1986.

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