MOLECULAR AND CELLULAR BIOLOGY, Sept. 1984, p. 1864-1870 Vol. 4, No. 90270-7306/84/091864-07$02.00/0Copyright © 1984, American Society for Microbiology

Cloning and Mapping of Saccharomyces cerevisiae PhotoreactivationGene PHRI

DAVID SCHILD,1* JOHN JOHNSTON,)+ CAREN CHANG,2t AND ROBERT K. MORTIMER1-

Division ofBiology and Medicine, Lawrence Berkeley Laboratory, 1 and Department of Biophysics and Medical Physics,2University of California, Berkeley, California 94720

Received 22 December 1983/Accepted 30 May 1984

The yeast Saccharomyces cerevisiae, like most organisms, is able to directly repair pyrimidine dimers byusing a photoreactivating enzyme and visible light. Cells carrying the phrl mutation were shown previously tobe unable to photoreactivate dimers, but neither the map position nor the primary gene product of the PHR1gene has been determined. We have cloned this gene and determined its map position. A plasmid containing a6.4-kilobase yeast DNA insert has been isolated and shown to restore photoreactivation in a phrl strain. A 3.1-kilobase subclone has also been shown to complement phrl. The original plasmid was targeted to integrate intochromosomal DNA at a site homologous to the insert by cutting within the insert. Two of these integrants havebeen mapped on the right arm of chromosome XV; the integrants have been further mapped at ca.13 centimorgans from prtl. It has also been independently determined that phrl maps at this location. Thus, wehave determined the map position of PHRI and also have shown that the plasmid contains PHRI rather than asuppressor of the phrl mutation.

The yeast Saccharomyces cerevisiae has several differentrepair mechanisms to deal with pyrimidine dimers caused byexposure to UV light (12, 18). Although most dimers areremoved by excision repair, they can also be repaired bydirectly splitting the dimers by photoreactivation. Photore-activation involves a photolyase (or photoreactivating en-zyme), a cofactor, and the absorption of visible light of lowwavelength (reviewed in reference 32). A yeast mutation(phrl) which is unable to photoreactivate dimers was isolat-ed by M. Resnick in 1969 (24, 25). Although the PHRI geneis clearly involved in photoreactivation, it is not known forwhich product the gene codes since (i) there appear to be twodifferent photoreactivating enzymes in commercial batchesof bakers' yeast, one a monomeric enzyme and the other aheterodimer, and each enzyme seems to require a cofactor(J. J. Madden and H. Werbin, personal communication), and(ii) a second photoreactivation-deficient mutation (phr2) hasrecently been isolated and shown to be loosely linked to phrl(19). To study the PHRI gene at a molecular level andeventually to determine its primary gene product, we havecloned this gene. A preliminary report on this cloning waspreviously presented (17). Yasui and Chevallier (33) haveindependently cloned a region which complements the phrlmutation (see below).Knowledge of the genetic map position of a gene is

becoming increasingly important for a full understanding ofthat gene. In the area of yeast recombinant DNA, geneticmapping has recently gained significance because plasmidintegration occurs via homologous recombination, and suchintegration events at the chromosomal location of a gene areconsidered strong evidence that a particular gene has beencloned (3, 13, 23). Since PHRI had not previously beenmapped, the cloned gene was used to determine the probablechromosomal location of PHRI, which would have been

* Corresponding author.t Present address: Department of Bioscience and Biotechnology,

University of Strathclyde, Glasgow, GI 1XW, Scotland.t Present address: Division of Biology, California Institute of

Technology, Pasadena, CA 91125.

extremely difficult to do by other means. The useful 2p.mintegrant mapping method of Falco and Botstein (10, 11) wasused to determine on which chromosome the PHRI-comple-menting fragment was located. Some difficulties with the2,um mapping method were encountered because of the useof the vector YEp13 (5); ways of avoiding these difficultiesare suggested (see below).

MATERIALS AND METHODSStrains and plasmids. The yeast strains are described in

Table 1. Escherichia coli strain HB101 (CA600 leu- B1-thr- pro- lacZ- Sm- recA- rB- mB- sufI) supplied by A. J.Clarke was used for bacterial transformation. A bank ofpartially Sau3A-digested yeast fragments cloned into theBamHI site of the vector YEp13 (5) was kindly supplied byK. Nasmyth (22).E. coli and yeast transformation. E. coli transformation

was performed as described by R. Davis et al. (8). Yeasttransformation was performed by a modified version of themethod of Hinnen et al. (13, 14) as previously described (29).Yeast transformants with replicating plasmids were distin-guished from revertants or plasmid integrants by streakingout for isolated colonies on nonselective medium (YEPD)and then replica plating to selective medium (in our case,synthetic complete medium without leucine). Transformantswith replicating plasmids frequently lose the plasmid onnonselective medium and subsequently do not grow onselective medium without leucine. Revertants and integra-tive transformants are stably Leu+ after growth on nonselec-tive medium.

Yeast genetic techniques and media. Tetrad analysis andyeast media were previously described (30). The canavanineploidy test (28) was used to determine the ploidy of transfor-mants; because canavanine resistance is recessive, haploidsmutate to resistance at a much higher frequency than dodiploids homozygous for canavanine sensitivity. Mitoticrecombination was performed by plating 100 to 500 cells perYEPD plate and treating with 10 krads of X ray to inducerecombination. The X-ray source was previously described(29). After colonies had formed, they were replica plated todrop out medium to allow detection of sectored or whole

1864

CLONING AND MAPPING A PHOTOREACTIVATION GENE 1865

TABLE 1. List of yeast strainsStrain Genotype" Source

PR74-35a a phrl-i ade2-1 his5-2 lelul-12 M. Resnicklysi-i meti-i trp548

X16-9C a rad2-1 ade2-1 Y.G.S.C.'DBY746 a his3-Al leu2-3 leu2-112 trpi-289 D. Botstein

ura3-52XS217-5B ot phrll-l rad2-1 leu2-3 leiu2-112 This studybDC04a a leu2-04 adel [cir°] J. Broach (5)XS95-6C a radS2-1 his3-Ai leu2-3 leui2-112 See reference 27

trpl-289 ura3-52 [cir°]XS84-38A a rad2 mesi adel ade3 Ieu2 h'sll This study

trpl ura3 hisXS445-1A a prtl leu2-3 leii2-112 ura3-52 This study

(iural?) his3-Al (his-?) ade2XS448-4C a leu2-3 leu2-112 met7 tIrp-289 This study

ura3-52XS476-9A a prtl phrl-i radl-l his3-Al ade2 This study

leu2 uralXS488-9B a [YEp13-PHR1-INT3] leu2-3 This study

leu2-112 rad2 ade2XS490-5D a [YEp13-PHRJ-INT4] leu2-3 This study

leli2-112 rad2 iuralXS500 ala [YEp13-PHR1-INT3]/+ leit2I This study

leu2 rad2lrad2 ade2l+ his3/+trplltrpl prtll+ phrll+ trpll+

XS502 a/oa [YEp13-PHRI-INT4]/+ leit2I This studyIeu2 trplltrpl prtll+ phrll+rad2lrad2 tvrll+

Most markers were derived from strains from the Yeast Genetic StockCenter (Y.G.S.C.), Department of Biophysics and Medical Physics. Universi-ty of California, Berkeley, Calif.

b Derived from the top three strains above.

colonies expressing recessive markers. Genetic analysis bythe 2,um mapping method is described in the text and isbased on work by Falco et al. (10, 11).

Screening for photoreactivation. LEU2 transformants fromregeneration agar plates were screened for photoreactivationby first inoculating them with sterile toothpicks into 0.2 ml ofmedium without leucine contained in wells of microtiterdishes (% wells per dish). PHRI RAD2, phrl RAD2, andphrl rad2 strains were also inoculated as controls. Microtiterdishes were incubated for a period of 18 to 24 h with slowshaking. Droplets were inoculated from these wells ontoagar plates by using a block holding a set of 48 solid-metalprobes. This procedure resulted in inocula of monolayers ofcells of fairly uniform cell density. These cells were irradiat-ed for 20 s with UV light from an 8-W General Electricgermicidal lamp emitting ca. 0.33 J/m2 per s. Immediatelyafter exposure, photoreactivating light was provided for 20min from two parallel 15-W General Electric blue-black lightbulbs (Fl5T8/BLB) at a distance of ca. 5 cm. Control plateswere immediately placed in the dark after UV exposure, andall plates were incubated in the dark for 2 to 3 days. Inoculawhich had undergone photoreactivation produced confluentgrowth, whereas inocula unable to carry out photoreactiva-tion produced only a few isolated colonies.

Photoreactivation of meiotic segregants from phrl hetero-zygotes was assayed as described above, except for use ofYEPD medium in microtiter wells, since these strains did notcontain any YEp13 (LEU2) plasmids. In later assays, anoth-er set of metal probes, in the form of retractable wire loops,was used to inoculate the plates (34). In addition, someassays were performed by lightly inoculating plates byreplica plating with Whatman no. 1 filter paper.

RESULTS

Cloning of PHRJ. Since photoreactivation can only bescored clearly in strains lacking excision repair, recipientstrains were constructed carrying both phrl and either of theexcision repair-deficient mutations radl or rad2 (12, 18). Offour closely related strains, only XS217-5B (Table 1) gavehigh transformation frequencies. Cold treatment for 1 h wasused during the transformation procedure (16). This treat-ment increased the frequency of transformants ca. 10-fold to-5,000 LEU2 transformants per ,g of DNA. A total of 1,560transformants of strain XS217-5B were screened for theirability to photoreactivate, and one presumptive PHRI clonewas recovered. Plasmid DNA was isolated from this cloneand used to transform E. coli strain HB101 to ampicillinresistance. Purified plasmid DNA from E. coli transformantswas then used to retransform strain XS217-5B. All of 43LEU2 transformants tested showed simultaneous transfor-mation for photoreactivation. Agarose gel electrophoresisafter restriction enzyme digestion of the purified YEp13-PHRI plasmid showed that the insert was ca. 6.4 kilobases(kb) and yielded the restriction map shown in Fig. 1. NoBamHI, HindIII, Sall, or Xhol sites were found in the insert.

Subcloning of PHRI. Subclones of the original 6.4-kbinsert were isolated to determine the location of the PHRIgene on the insert and to develop a more detailed restrictionmap of the PHRI region. An -3.1-kb PvuII fragment wassubcloned in both orientations into the YRp7 yeast replicat-ing vector (3) which contains the yeast TRPI gene (Fig. 2).Both of these subclones (YRp7-PHRI-A and YRp7-PHRI-B) complemented the phrl mutation, indicating that both thecomplete structural gene and its promotor are probablylocated in this 3.1-kb region. Subclones YRp7-PHRI-C andYRp7-PHRI-D were constructed by deleting a short Clalfragment from YRp7-PHRI-A and YRp7-PHRI-B, respec-tively (Fig. 2). Neither of these subclones complemented thephrl mutation, indicating that the PHRI gene probablystraddles this Clal site. This Clal site could therefore beused for gene disruption experiments and as a site to startsequencing the PHRI gene. The subclones, because of thissmall size, have also simplified construction of a moredetailed restriction map of this area. In addition to confirm-ing sites found in YEp13-PHRI, cleavage sites for AvaI,Clal, NcoI, NdeI, and XbaI were found and mapped in this3.1-kb region (Fig. 2). No NruI, Sacl, SmaI, SstII, or StuIsites were found in this region.

Survival curves. Survival curves were used to confirm theresults from plate assays that the YEp13-PHRI plasmidcomplemented the phrl mutation. To interpret the survivalcurves, it was necessary to know the ploidy of the yeasttransformant used. Our original PHRI transformant andabout half of our retransformed cells were shown to bediploid or of higher ploidy, presumably due to spheroplastfusion during the transformation procedure. The transfor-mant used for the survival-curve experiment was shown tobe a haploid strain by both the canavanine ploidy test (28)and by tetrad analysis of a diploid constructed by mating thetransformant to a known haploid. Survival curves wereperformed on stationary-phase cells since it has been shownin S. cerevisiae that stationary-phase cells have much higherlevels of photoreactivation than logarithmically growingcells (2). Also, since replicating plasmids are mitoticallyunstable in S. cerevisiae (3), growth of cells and platings forthe survival curves was done on synthetic complete mediumwithout leucine for strains with the YEp13-PHRJ (LEU2)plasmid. Strains not containing a plasmid were plated on

VOL. 4, 1984

1866 SCHILD ET AL.

Sa II

Yeast chromosomal DNAZ.,//Z 2 DNA

pBR3221 kb

--I

*Bgl II

Pst I

'HRI Insert=6.4kb)

IN

"II.N\N\

aN

IIPstI BgIII PvuIW(BomHI/Sau3A)

BgII EcoRIl PvuII Bgl l|EcoRI BglU (BamHI/Sau3A)

FIG. 1. Restriction map of the YEp13-PHRJ plasmid. Underlined PvuII sites were used in the targeting experiment. (BamHI-Sau3A) sitesare not cut by BamHI.

synthetic complete medium. After UV irradiation, cells to bephotoreactivated were given 30 min of photoreactivatinglight; this exposure was shown to give maximal photoreacti-vation for the strains used. The survival curves (Fig. 3)demonstrate that the rad2 phrl strain showed increasedsurvival after UV and photoreactivating light only when thecells contained the plasmid. The rad2 PHRI strain and therad2 phrl strain containing the plasmid both had very similarsurvival curves, indicating that the plasmid restored fullphotoreactivating ability. These survival curves can not beused to quantify the amount of photolyase in these cells,since the cells were given 30 min of photoreactivating light,and photolyase recycles during photoreactivation (32).

Targeted integration of YEp13-PIIR1 and mapping theintegration site. Plasmid integration into yeast chromosomeshas been shown to occur via recombination between yeastsequences on the plasmid and homologous sequences inchromosomal DNA (13, 23). Therefore, to determine thechromosomal region contained in the insert of YEp13-PHRI, we isolated cells in which integration events hadoccurred, and the site of integration was genetically mapped.Since spontaneous integration events of the plasmid couldand probably did occur at many sites in the genome becauseof a partial TY1 sequence carried on the vector YEp13 (9)(see below), we also isolated targeted integrants. Targetingrefers to cutting a plasmid with a restriction enzyme beforeyeast transformation. This usually results in the integrationof the plasmid into a chromosomal region homologous tothat region which was cut on the plasmid (13, 23). Ideally,targeting is initiated by restricting the plasmid with anenzyme which only cuts within the region of interest. Sincenone of the characterized restriction sites in our plasmidappeared uniquely in the cloned insert, plasmid DNA waspartially restricted with PvuII before transformation of theleu2 strain DC04a (Table 1). PvuII cuts the plasmid twice inthe insert and once in pBR322 sequences (Fig. 1). Transfor-mation with the partially digested DNA resulted primarily intransformants containing circular plasmid DNA since theplasmid contains a yeast origin of replication and since such

replicating circular plasmids transform with much higherefficiency than do linearized plasmids (3, 23). Transformantswith replicating plasmids are easy to recognize since they areunstable on nonselective growth medium (see above). Twostable Leu+ colonies were found among the 30 presumptivetransformants tested, although these could have been eitherintegrated transformants or LEU2 revertants, since the leu2marker in DC04a is revertable at a low frequency (4).To determine simultaneously whether these were rever-

tants or integrated transformants and, if the latter, intowhich chromosome the plasmid had integrated, the methodof Falco et al. (10, 11) of 2,um integrant mapping was used.This method is based on observations with integrated plas-mids containing one or both of the inverted-repeat sequencesof the yeast-endogenous 2,um plasmid. When such integrantsare present in a [cir+] diploid (a diploid containing endoge-nous 2,um DNA), genetic markers on both the integratedplasmid and on the chromosome into which the plasmid hasintegrated are lost at a high frequency (up to 50%) (10, 11,15). Most of these events can not be explained by simpleexcision of the integrated plasmid and may occur by anunequal sister chromatid exchange and associated events(11, 15). The two presumptive transformants resulting fromintegration events were each crossed to Ieu2 strains so that,if they were transformants, they would be homozygous forleu2 on chromosome III but phenotypically Leu+ because ofthe LEU2 gene on the integrated plasmid. Therefore, loss ofthe LEU2-integrated gene should result in Leu- sectors orcolonies. The two presumptive transformants (DC04a-INT2and -INT3) were crossed to three different leu2 strains, andtwo of the three sets of crosses showed the high frequency(-20 to 30%) of leu2 segregants expected for integratedtransformants containing one of the inverted repeats of 2,umDNA. This demonstrated that the original two stable Leu+colonies were actually integrated transformants and notrevertants. Fortuitously, among these first crosses werecrosses to strain DBY746 (a leu2 his3 ura3 trpl [cir+]), andthese diploids also sometimes expressed the his3 marker, butat a lower frequency (-5 to 10%) than leu2. Since the HIS3

MOL. CELL. BIOL.

to

CLONING AND MAPPING A PHOTOREACTIVATION GENE 1867

YRp 7-PHR1-8 (Insert=3.1 kb)

BglI11 EcoRIXoI ClaIlvu X " I II I'

k

NdeIbaI Ava I \ Bgl I

NdeI EcoR I

PHR1 Activity

YRp7-PHRI-C (Insert=1.5kb)

Bgl UI

YRp7-PHRI -D (insertz1.6kb)

XbaI NdeEcoRI EcoRI(Nru I/Pvu U)

iaI\ NcoI Clol

Bgl E NdeI

FIG. 2. Subclones in YRp7 with and without PHRI activity. The inserts in YRp7-PHRI-A and -B are identical but in opposite orientation.The YRp7-PHRI-C plasmid was derived from YRp7-PHR1-A by deletion of the short ClaI fragment (ClaI sites are underlined); YRp7-PHRI-D was derived in a similar fashion from YRp7-PHR1-B. (NruIlPi'uII) sites are not cut by either enzyme. The order of other restriction siteswithin parentheses is not known.

gene is on chromosome XV and sometimes was lost concur-rently with the integrated plasmid, this result indicates thatthe integration event had occurred on chromosome XV.The set of crosses which did not show high levels of leu2

segregants were the crosses of the two integrants by XS95-6C (ot radS2-1 Ieu2 his3 ura3 trpl ). The most probableexplanation for this was that XS95-6C is [cir°], since Falco etal. (11) have shown that diploids lacking endogenous 2,umDNA do not undergo the marker loss associated withintegrated 2,um plasmids. Since one parent in each of ourcrosses (DC04a-INT2 and DC04a-INT3) is known to be[cir°], crosses to another [cir°] strain would result in [cir°]diploids. Although most laboratory strains of S. cerevisiaeare [cir+], we have recently determined by Southern analy-sis that XS95-6C is in fact [cir°], as are several of our otherrad52 mutant strains, and we are currently investigatingwhether the radS2 mutation generally causes loss of 2,umDNA. The stability of the markers in these [ciro] diploidsallowed us to perform conventional mitotic recombination

analyses which would have been virtually impossible in the[cir+] diploids because of the instability of markers. Mitoticrecombination induced by X rays (see above) in the diploidconstructed with integrant 1 resulted in 12 Leu- His'colonies (or sectored colonies), 6 Leu- His- colonies, and 1Leu+ His- colony. Similarly, the diploid with integrant 2gave six Leu- His' colonies, seven Leu- His- colonies, andone Leu+ His- colony. These results indicated that theplasmid in both transformants had integrated on the rightarm of chromosome XV, distal to his3 (Fig. 4).

Tetrad analysis was used to confirm assignment of theintegration events to the right arm of chromosome XV and todetermine the location of the integrants on this chromosome.Since his3, met7, and prtl are located on the right arm ofchromosome XV (Fig. 4), the two integrants were eachcrossed with his3 leu2, met7 Ieu2, and prtl leu2 strains(Table 1). Tetrad analysis showed that in both cases theintegrated LEU2 gene was linked to prtl but not to his3 ormet7 (Table 2).

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1868 SCHILD ET AL.

0

0uInto

0

z

La

0

a.

3 4

U.V. (J/M2)

FIG. 3. Survival curves. Continuous lines indicate that UV irra-diation was followed by incubation with photoreactivating light, anddashed lines indicate no photoreactivating light.

x3v

iade2-SUF5

, Lserl_ ,ade9

This3(his8)

cdc3lmet7tra3

cpal_ suf 13

-MAL1XSUC'1

- fol2- mesl

- rad2SF5INTI

ADE15

ade3-frol

- SUP76

-fro2

SUP77SUF4

-prt 1

FIG. 4. Map position of phrl and one of the integration sites ofYEp13-PHRJ. Only parts of the right arms of chromosome VII andXV are shown; arrows point towards the centromere.

TABLE 2. Tetrad analysis data for two integrated PHRIplasmids and for the phri mutation

Intervala Ascustype' Map distancePD NPD T (cM)"

[YEp13-PHR1-INT2]-prtl 31 0 12 14.0 ± 3.5[YEp13-PHRI-INT3]-prtl 12 0 2 7.4 + 5.1[YEp13-PHR1-INT3]-met7 4 2 11 N.L.[YEp13-PHR1-INT3]-his3 8 6 16 N. L.phrl-prtl 43 0 16 13.6 ± 2.9[YEp13-PHR1-INT2]-phrl 7 0 0 0[YEp13-PHRl-1NT3]-phrl 14 0 0 0

a Mapping data from two or more similar crosses were sometimes com-bined, but data from cross to cross were similar.

b PD, Parental ditype; NPD, nonparental ditype; T, tetratype.' Map distances and standard error were determined by the mapping

functions of R. Snow (31). N.L., Not linked; cM, centimorgans.

Diploids heterozygous for the integrated plasmids andprtl, in coupling, were also constructed and found to sponta-neously segregate both Leu- Prt+ and Leu- Prt- colonies.Of a total of 301 colonies from diploid XS500 (heterozygousfor plasmid integrant 1 [see Table 1]), 39 Leu- Prt+ (13%)and 9 Leu- Prt- (3%) colonies were observed. Similarly, 40Leu- Prt+ (10%) and 8 Leu- Prt- (2%) colonies wereobserved out of 409 colonies from XS502 (heterozygous forintegrant 2). Falco et al. (10, 11) normally observed thatmarkers distal to integration events of 2pum plasmids werealmost always coordinately expressed when plasmid mark-ers were lost. Since this was not observed in the diploidsconstructed, we interpret our results to indicate that theplasmid had probably integrated distal to prtl.

Determining the map position of PHR1. From the afore-mentioned experiments, it is clear that the plasmid weisolated complements phrl-l and integrates near prtl onchromosome XV. To show that the plasmid contains se-quences homologous to PHRI, rather than to some type ofsuppressor of phrl, it was necessary to show that PHRImapped at the same location as the plasmid integrated. Thegenetic map position of PHRI has not previously beendetermined. The phrl mutation can be easily scored only inan excision-deficient background, such as rad2, thus makingthis gene difficult to map by most traditional methods. Oncea possible site for PHRI was found by the mapping of theplasmid integrants, it was relatively easy to determinewhether PHRI actually did map near PRTI on the right armof chromosome XV. Diploid strains were constructed whichwere homozygous for rad2 and heterozygous for both phrland prtl. Tetrad analysis of these diploids revealed that phrldid in fact map near prtl and at a distance at or near the sitewhere the plasmid had integrated (Table 2; Fig. 4). Analysisof crosses of the phrl prtl rad2 leu2 haploid XS476-9A(Table 1) with rad2 leu2 strains carrying the integratedplasmids (XS488-9B and XS490-5D [Table 1]) demonstratedthat phrl and the LEU2 gene on the integrated plasmidalways segregated as parental ditypes (Table 2). This strong-ly indicates that the map position of PHRI and the integra-tion site of the plasmid are identical. Since prtl is quite farfrom met7 (7, 21), the nearest easily scorable marker, theside of prtl on which the phrl gene is located has not beendetermined with certainty by tetrad analysis. Brackets havebeen placed around prtl and phrl (Fig. 4) indicating thatphrl might be centromere proximal to prtl, although experi-ments presented above indicate that the plasmid integrantand therefore phrl also, probably map centromere distal toprtl.

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CLONING AND MAPPING A PHOTOREACTIVATION GENE 1869

Incidental mapping of a possible TY1 or 8 chromosomalsite. While attempting to locate the chromosomal site ho-mologous to the insert in the YEp13-PHRI plasmid, weincidentally located a possible chromosomal site of a TY1 orsolo 8 sequence. TY1 is a yeast-transposable element, and asolo 8 sequence is a single copy of the sequence which is alsofound as an inverted repeat at the ends of TY1 sequences.Since our first attempt at targeting integration by using aPvuII partial digest was unsuccessful and since we wereunaware at the time that the YEp13 vector contained part ofa TY1-17 sequence (9), spontaneous integration events ofreplicating plasmids were isolated with the intent of mappingthe chromosomal site of the PHRJ insert. One spontaneousintegration event into strain DC04a (Table 1) was deter-mined, by the 2,um mapping method of Falco et al. (10, 11)described above, to have occurred on chromosome VII;tetrad analysis of a cross between this integrant and the ade3rad2 mesi leu2 strain XS84-38A (Table 1) showed that theintegration event had occurred between ADE3 and RAD2(Table 3; Fig. 4). However, tetrad analysis of crossesbetween phrl strains and strains carrying the ade3, rad2, andmesl markers yielded no evidence of linkage between phrland these genes (data not shown). Since our results indicatethat the insert in plasmid YEp13-PHRI does contain PHRIand that PHRI does not map at this location, the most likelyexplanation is that the plasmid had integrated at a TY1 orsolo 8 sequence (see below).

DISCUSSIONTo study at the molecular level yeast genes involved in

DNA repair, we have cloned several of these genes (6, 29)and here report on the cloning of a yeast gene (PHRJ)involved in photoreactivation repair of pyrimidine dimers.Similar cloning of E. coli repair genes (reviewed in reference26), including a photoreactivation gene (27), has been ex-tremely useful in elucidating the exact role of these genes inDNA repair. The cloned yeast PHRI gene should be usefulin determining the protein for which this gene codes. It is notclear whether PHRI codes for the yeast photolyase (if it is amonomeric enzyme) or for one of the subunits of a dimericenzyme. It is even possible that PHRI codes for a cofactorinvolved in photoreactivation or a regulator of photolyasesynthesis or activation. Yasui and Chevallier (33) haveindependently cloned a yeast DNA fragment which comple-ments the phrl-J mutation. When present on a high-copy-number plasmid in photoreactivation-proficient S. cerevisiaecells, this cloned fragment results in a 15-fold increase in thenumber of photolyase molecules per cell (33). Although thisis evidence that the plasmid of Yasui and Chevallier may

TABLE 3. Genetic mapping of a plasmid integrant at a probableTY1 or solo 8 sequence"

Intervalb Ascus type Map distancePD NPD T (cM)

[YEp13-PHRJ-INT1]-ade3 19 0 3 6.9 ± 3.9[YEp13-PHRJ-INT1]-rad2 17 0 6 13.2 + 4.7[YEpl3-PHR1-INTl]-mesl 11 0 10 23.9 ± 5.6rad2-ade3 12 0 10 22.8 ± 5.5rad2-mesl 17 0 5 11.5 ± 4.7mesl-ade3 8 0 14 31.9 + 5.3

" PD, Parental ditype; NPD, nonparental ditype; T, tetratype; cM. centi-morgan.

b Mapping data from two or more similar crosses were sometimes com-bined, but data from cross to cross were similar.

contain the PHRI gene and that this gene codes for the yeastphotolyase, other interpretations are possible. Their plasmidcould contain a positive regulator of PHRJ with phrl havingpartial photolyase activity. Alternatively, they may havecloned the actual PHRI gene, but PHRI itself may code foran inducer of the real photolyase gene or genes.

Since plasmid integration in S. cerevisiae has been shownto occur by homologous recombination (13, 23), the mostconclusive evidence that a particular yeast gene has beencloned is to integrate it into the genome and show that theintegrated plasmid maps at the chromosomal location of thegene being cloned (3). Integrants of our plasmid have beenisolated and found to map at the chromosomal location ofPHRI, proving that our plasmid contains the PHRI gene.The restriction pattern of our insert appears very similar tothat of the insert isolated by Yasui and Chevallier (33), andthis is supported by the fact that -3-kb PvilII subclones fromeach insert complement phrl. Because of these similarities,it seems likely that these two plasmids contain homologoussequences and that the plasmid isolated by Yasui andChevallier actually does contain the PHRI gene.

Since the genetic map position ofPHRI had not previous-ly been determined, it was necessary to map this gene toshow that the plasmid integration events had occurred at thesite of PHRI. Since the genetic map of S. cerevisiae isextremely long and divided into many linkage groups (20)and since the phrl-l mutation is rather laborious to score,the chromosomal location of PHRI would have been ex-tremely difficult to obtain by traditional genetic methods. Incontrast, the integrated plasmid thought to contain PHRIalso contained the LEU2 gene, which is very easy to score.By first using a new mapping method (10, 11) specific forintegrated plasmids containing part of the yeast 2,um plas-mid, followed by both mitotic recombination and tetradanalysis, the integration events were located on the right armof chromosome XV. It was then possible to show that PHR1did actually map at this location. In the process of using the2,um mapping method (10, 11), we encountered two difficul-ties which should be avoided. The PHRJ clone was isolatedfrom a frequently used yeast bank constructed by K. Na-smyth (22). This bank was made in the yeast vector YEp13(5). Since the YEp13 vector contains both part of a yeastTY1-17 transposable element (9) and a leucine tRNA gene(1), it has the potential to integrate at many sites in the yeastgenome. One spontaneous integration event of our YEp13-PHRI plasmid occurred on chromosome VII, although ourinsert does not actually map at this site. Although it ispossible that this site on chromosome VII contains a regionwith some homology to our PHRI insert, the most likelyexplanation is that this is the site of a TYl-like sequence or asolo 8 sequence, one of the sequences normally found as arepeat at the ends of a TY1 element. Two methods ofavoiding integration events at LEU2, TY1 sequences, solo 8sequences, or leucine tRNA genes are to either target theintegration of the plasmid by restricting it in the insertedregion (13, 23) or to subclone into the YEp24 vector (3),which has the URA3 gene in place of the LEU2 gene ofYEp13. A second problem we encountered with the 2,ummapping method was the lack of chromosome instability of a[ciro] diploid (see above). This problem can be avoided byeither integrating the 2,um plasmid into a [cir+] strain ormaking sure that the tester strains with the various chromo-somes marked are [cir+] strains. Since most yeast strains are[cir+], this should not normally be a problem.The cloned PHRJ gene should be useful in determining

whether this gene actually codes for a yeast photolyase and

VOL. 4, 1984

1870 SCHILD ET AL.

whether this enzyme is monomeric or dimeric. The clonedgene could also be used for gene regulation studies. It hasalready proven valuable in determining the map position ofPHRI, as reported here.

ACKNOWLEDGMENTS

We thank Chris Alafi, Noel Fong, Ken Mason, and TommyMcKey for excellent technical assistance and Diana Morris for helpwith this manuscript.

This work was supported by a grant from the Cancer ResearchCoordinating Committee of the University of California, PublicHealth Service grant GM 30990 from the National Institutes ofHealth, and a grant from the Office of Health and EnvironmentalResearch of the U.S. Department of Energy under contract numberDE-AC03-76SF00098. J. Johnston acknowledges provision of travelgrants by the Royal Society and the University of Strathclyde.

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