MOLECULAR AND CELLULAR BIOLOGY, Aug. 1990, p. 3917-3925

Vol. 10, No. 8

0270-7306/90/083917-09$02.00/0 Copyright © 1990, American Society for Microbiology

Mutational Analysis of the Consensus Sequence of a Replication Origin from Yeast Chromosome III J. VIRGINIA VAN HOUTEN AND CAROL S. NEWLON* Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103 Received 7 February 1990/Accepted 27 April 1990

Yeast autonomously replicating sequence (ARS) elements contain an 11-base-pair core consensus sequence

(5'-[A/T]ITTTAT[A/GJTTT[A/T]-3') that is required for function. The contribution of each position within this sequence to ARS activity was tested by creating all possible single-base mutations within the core consensus sequence of ARS307 (formerly called the C2G1 ARS) and testing their effects on high-frequency transformation and on plasmid stability. Of the 33 mutations, 22 abolished ARS function as measured by hig-frequency transformation, 7 caused more than twofold reductions in plasmid stability, and 4 had no effect on plasmid stability. Mutations that reduced or abolished ARS activity occurred at each position in the consensus sequence, demonstrating that each position of this sequence contributes to ARS function. Of the four mutations that had no effect on ARS activity, three created alternative perfect matches to the core consensus sequence, demonstrating that the alternate bases allowed by the consensus sequence are, indeed, interchangeable. In addition, a change from T to C at position 6 did not perturb wild-type efficiency. To test whether the essential region extends beyond the 11-base-pair consensus sequence, the effects on plasmid stability of point mutations one base 3' to the T-rich strand of the core consensus sequence (position 12) and deletion mutations that altered bases 5' to the T-rich strand of the core consensus sequence were examined. An A at position 12 or the removal of three T residues 5' to the core consensus sequence severely diminished ARS efficiency, showing that the region required for full ARS efficiency extends beyond the core consensus sequence in both directions.

Autonomously replicating sequence (ARS) elements of Saccharomyces cerevisiae were identified on the basis of their ability to promote extrachromosomal maintenance of plasmid DNA in cis (19, 42). Direct examination of replicating DNA has revealed that ARSI and the 2ixm ARS function as replication origins on plasmids (7, 20) and that at least some ARS elements are active as chromosomal origins of replication (21, 26a; S. Greenfeder and C. S. Newlon, unpublished data). Mutational analysis of several ARS elements shows that they contain an essential region which has been called the core (4, 5, 9, 24, 39) or domain A (9, 39). This region contains a sequence of at least 11 base pairs (bp), whose consensus is 5'-(A/T)T1TT-AT(A/G)TllT(A/T)-3' (8, 41). Mutations, small deletions, and linker substitutions that occur within or include part of the core consensus sequence abolish or severely reduce ARS activity, demonstrating that the core consensus sequence is essential for ARS function (4, 6, 9, 24, 33). Although mutations in the core consensus sequence have been shown to affect ARS activity severely, many ARS elements contain only a single imperfect match to the consensus sequence (1, 15, 23, 24, 37, 41). Thus, whereas deviations from the core consensus sequence at some positions of this critical region abolish ARS activity, mutations at other positions do not. To investigate the importance of each base within the core consensus sequence, all possible point mutations within the perfect match to the consensus sequence contained in the chromosome III ARS element, ARS307 (formerly called the C2G1 ARS [33, 34]) were created. ARS307 functions as a chromosomal origin of replication (Greenfeder and Newlon, unpublished data), and its perfect match to the core consen* Corresponding author.

sus sequence has been shown to be essential for function by deletion analysis (33).

MATERUILS AND METHODS Bacterial and yeast strains. Escherichia coli JA226 (14) was used as the host strain for plasmid DNA isolation. E. coli TG1 (46) was used for all M13 constructions and isolations. S. cerevisiae YNN216 (36) MATa/MAToa ura3-521ura3-52 lys2-8011lys2-801 ade2-101/ade2-101 was used to test ARS phenotypes of all plasmid pVHA constructs. The M13mpl8 bacteriophage vector used to isolate the ARS307 deletion fragments was obtained from Bio-Rad Laboratories. The M13mp8 vector used to isolate all core mutations was a gift from Zafri Humayun. Media and reagents. JA226 transformants (11) were grown in Luria broth (30) supplemented with 50 ,ug of ampicillin per ml. E. coli TG1 was maintained on glucose minimal medium plates (27) and was grown in 2xYT medium for transfection and DNA isolation (30). Yeast strain YNN216 was maintained on YEPD plates (35). -Ura medium contained 6.7 g of yeast nitrogen base without amino acids, 10 g of Casamino Acids (Difco Laboratories), 10 mg of adenine, 10 mg of tryptophan, and 20 g of glucose per liter. For plates, 20 g of Difco agar per liter was added. Color assay medium contained 6.7 g of yeast nitrogen base, 5 g of Casamino Acids, 20 mg of uracil, 4.5 mg of adenine, 10 mg of tryptophan, 20 g of glucose, and 20 g of agar per liter. Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, and restriction enzyme linkers were obtained from New England BioLabs, Inc. DNA polymerase I (Klenow fragment) and calf intestinal alkaline phosphatase were obtained from Boehringer Mannheim Biochemicals. a-32p_ labeled deoxyribonucleotides and the oligonucleotidedirected mutagenesis kit were purchased from Amersham

Corp. 3917

3918

VAN HOUTEN AND NEWLON A.

MOL. CELL. BIOL.

ARS307 SEQUENCE 10 30 40 50 20 GAATTCTAGG TGATATTGCA ATTACTTCTT CTCATGCACT AACATGTGAA CTTAAGATCC ACTATAACGT TAATGAAGAA GAGTACGTGA TTGTACACTT

60 80 90 100 70 TGATAGAAAT ATGTTGAGTT CCTAACTGCC TGAT T AAA TAAGTMTCAT ACTATCTTTA TACMCTCM GGATTGACGG ACTAAAATTT ATTCAAAGTA 110 130 140 150 120 ATTATAATCT TTTAGCATAT ATATATATAT ATTGATCCTC TCTCTTCTTT TAATATTAGA AAATCGTATA TATATATATA TAACTAGGAG AGAGAAGAAA 160 170 180 190 200 ATTTTCTGCC AGTAACCCAT GTGTGAAGAA GAAAACATAA ATAAAAAAGC TAAAAGACGG TCATTGGGTA CACACTTCTT CTTTTGTATT TATTTTTTCG ..ee........

B.

PLASMID

SEQUENCE

RATE OF LOSS

pVH400/437

160 169 ...CTGCC AGTMCCCAg GcaTGc ...GACGG TCATTGGGTc CgtACg

Ars-

pVH402

160 180 190 170 CTGCC AGTAACCCAg GcaTGcAGAA GAAAACATAA ATAAAgtcga c ... GTCGG TCATTGGGTc gcac g

.235+.016

pVH440

...

... ...

160 170 180 190 200 CTGCC AGTAACCCAT GTGTGAAGAA GAAAACATAA ATAAAAAAGC ggcatgc GACGG TCATTGGGTA CACACTTCTT CTTTTGTATT TATTTTTTCG ccgtacg

.200+.006

see*00 *O*@*

FIG. 1. Construction of semisynthetic ARS307 for mutagenic analysis. (A) DNA sequence of bases 1 to 200 of ARS307 as published by Palzkill and Newlon (33) and amended at base 71. This fragment was shown to have optimal ARS activity (33). The perfect match to the T-rich strand of the core consensus sequence is underlined. The overlapping near match to the core consensus sequence is shown by dots. Since the T-rich strand of each of these consensus sequences is in the lower strand, the 3'-flanking region is to the left and the 5'-flanking region is to the right. (B) DNA sequences of bases 155 through the end of the 169-bp deletion fragment, the semisynthetic construct, and the wild-type 200-bp deletion fragment. All constructs contain sequences beginning with position 1 in panel A. SphI and Sail sites are shown in italics. The 29-base single-stranded oligonucleotide added to the 169-bp deletion fragment is boxed. Changes from the ARS307 sequence are shown in lowercase letters. Plasmid numbers refer to the pVHA plasmid carrying the ARS constructs shown. Loss rates were calculated as described in Materials and Methods and represent the mean and standard deviation of measurements of at least three independent transformants.

Oligonucleotides used for cloning and for site-directed mutagenesis were synthesized on an Applied Biosystems 380A oligonucleotide synthesizer. Plasmid isolation. Plasmid DNA was isolated from E. coli by the alkaline lysis method (27) and was purified through a cesium chloride step gradient (16). Small-scale plasmid preparations were made according to the alkaline lysis procedure of Birnboim and Doly (3). Vectors. Plasmid pVHA is a YIp5 (45) derivative. It was constructed by attaching SalI linkers at the YIp5 PvuII site and inserting a 1.4-kilobase (kb) XhoI fragment of CEN4 (28) at the SalI linker. A 0.72-kb HaeII-BclI fragment of SUPJJ (43), to which BamHI linkers had been attached, was inserted into the BglII site of the CEN4 fragment. CEN4 is oriented within this fragment so that element III is closest to the tetracycline resistance gene of plasmid YIp5. SUPJJ is oriented such that transcription begins on the oriC side of the plasmid and proceeds towards CEN4. Deletion fragment isolation. ARS307 deletion fragments were identified by screening a Bal 31 deletion library constructed by Palzkill and Newlon (33). Before construction of the library, a BamHI linker had been attached at position 1 of the ARS307 sequence (Fig. 1), and SphI linkers were

added at the deletion endpoints. These BamHI-SphI fragments were removed from the parent plasmid, separated in 6% acrylamide gels, and electroeluted. The AR5307 deletion fragments were then ligated to M13mpl8 and sequenced. To test all BamHI-SphI deletion fragments in the same vector context as that of the semisynthetic AR5307 constructs, the deletion fragments were placed between BamHI and SalI in plasmid pVHA (plasmids pVH437, pVH440, pVH441, and pVH442). To do this, the M13mpl8 vector containing the deletion fragments was digested with HindIll, the ends were filled in with DNA polymerase (Klenow fragment), and SalI linkers were attached. The resulting BamHI-to-Sall fragment was then excised from M13mpl8 and ligated to plasmid pVHA which had been cut with BamHI and Sail. This manipulation resulted in 5 bp of the polylinker region of M13mpl8 being appended to the SphI end of the deletion fragments. ARS307 core mutation constructions. Synthetic oligonucleotides were phosphorylated with T4 polynucleotide kinase (27) and were cloned according to the procedure of Derbyshire et al. (13), which allows cloning of single-stranded oligonucleotides. Briefly, the vector was first cleaved with Sall (creating a 5' overhang), treated with calf intestinal

MUTATIONAL ANALYSIS OF AN ARS CORE CONSENSUS SEQUENCE

VOL. 10, 1990

3919

0

I.-

sv o

0

0

-.5

0

0

-i

C)

0

m

0 0

0

0

0

0

-1 0

0 0 -J

a

-1.5

a

.1

.2

.3

.4

.5

.6

RATE OF LOSS FIG. 2. Correlation of mitotic stability to rate of loss. A series of experiments was performed to establish the correspondence between the mitotic stability of representative semisynthetic ARS307 plasmids and their rate of loss when selection was relieved. In these experiments, cultures were grown under selection in -Ura medium. At zero time, a sample was plated on -Ura and color assay media to determine the fraction of the population containing plasmid (mitotic stability), and selection on the culture was relieved by adding uracil. Samples of the culture were plated on -Ura and color assay media at subsequent time points. Loss rates were calculated from the slope of the straight line resulting from plotting log plasmid containing cells against generations after relief of selection. A regression line correlating the log of the mitotic stability of these plasmids to their measured rates of loss under nonselective conditions was constructed. Each point on the figure represents the measured loss rate and mitotic stability of a single transformant. The plasmids used to construct this regression line ranged in loss rates between 0.115 and 0.579 per generation. By using this line, which had a correlation coefficient of -0.85, the rate of loss of each plasmid could be calculated after determining its mitotic stability.

alkaline phosphatase as described by the manufacturer, gel purified, and electroeluted. A 100-ng sample of treated vector was then ligated with a 104 molar excess of kinased oligonucleotide for 4 h at 16°C. This ligation mixture was heat inactivated at 65°C for 10 min and digested for 1.5 h with SphI (producing a 3' overhang). The restriction enzyme was heat inactivated, and the mixture was diluted 25-fold in ligation buffer. DNA ligase was added, and the ligation was allowed to proceed overnight at 16°C. The ligation mixture was then used to transform E. coli. Because of restriction site limitations, the construction containing the wild-type core oligonucleotide (pVH402) was created in plasmid pVHA, which contained the ARS307 169-bp deletion fragment between BamHI and SphI (pVH400) (Fig. 1). The semisynthetic ARS307 fragment (169:01) was then cloned into M13mp8 between BamHI and SalI (Ml3mp8:169:01), and its DNA sequence was determined. To make mutations in the 11 bases of the core consensus sequence and at position 12, 12 oligonucleotides were synthesized, each with an equimolar mixture of the three alternate bases at a single position. The mutant oligonucleotides were used to replace the wild-type oligonucleotide in M13mp8:169:01 as described above. Site-directed mutagenesis. A 2-bp mutation in the core consensus sequence of the semisynthetic ARS307 construct that corresponds to the 522 CN6 mutation created by Palzkill and Newlon (33) was generated in M13mp8:169:01, using a kit purchased from Amersham as instructed by the manufacturer. ARS phenotype determination. Yeast strain YNN216 was transformed by the method of Ito et al. (22), and Ura+ transformants were picked and streaked on -Ura plates. Colonies arising on these plates were subcultured again on

-Ura plates. Plasmids giving rise to transformants that could persist through two subcultures on selective medium were scored as Ars+. All others were scored as Ars-. Plasmid stability. Plasmid loss rates (per generation) were determined from the fraction of plasmid-containing cells under selective conditions (mitotic stability) as described by Palzkill and Newlon (33). Briefly, a regression line (method of least squares) was constructed to relate mitotic stability with loss rates determined under nonselective conditions (Fig. 2). All loss rates reported in this paper were calculated from the mitotic stability data by using the regression line and reflect the average values obtained from at least three independent transformants.

RESULTS Test plasmid construction. Plasmid pVHA was constructed to make use of the colony color assay system of Hieter et al. (17) to determine ARS efficiency. This system depends on the suppression of an ade2-ochre mutation by a plasmidborne ochre-suppressor tRNATYr, SUPJJ (43). The 1.35-kb EcoRI SUPIJ fragment previously used in test plasmids also contained ARS3 (43). Although ARS3 is a "weak" ARS (17), these plasmids are nevertheless able to transform at high frequency and to persist in a low proportion of the population. For the purposes of this study, it was imperative that no contaminating ARS function be present on the vector. To construct the test plasmid, pVHA, a 0.72-kb HaeII to Bcul fragment containing SUPJI that is devoid of any detectable ARS activity was used. In addition to SUPJJ, plasmid pVHA also carries URA3 and CEN4. When plasmid pVHA is used to transform yeast strain YNN216, it produces abortive transformants similar to those

3920

VAN HOUTEN AND NEWLON

formed by plasmid YIp5, which are unable to persist upon subculturing. This vector can be used to determine unambiguously whether a mutant ARS element is functional as assayed by its ability to promote high frequency transformation. For mutants that are Ars+, the plasmid can be used to measure accurately even weak ARS function in plasmid stability assays. Rates of loss measured for plasmid pVHA containing ARSI (0.044 + 0.003) are similar to those noted by others (12, 17, 26, 44). Therefore, measurements of ARS efficiency made in the plasmid pVHA vector can be compared with the efficiencies reported for other ARS elements in CEN-containing vectors. Experimental design. To determine the nucleotide sequence requirements for an ARS core consensus sequence, we wanted to study an ARS element with the following properties. First, it should contain an exact match to the core consensus sequence that had been shown to be essential for ARS function. Second, it should be a moderate to weak ARS rather than a strong ARS so that mutations that increased ARS efficiency as well as mutations that decreased ARS efficiency would be detectable. ARS307 (formerly C2G1) has both of these properties (Fig. 1). It is the only one of four chromosome III ARS elements sequenced by Palzkill et al. (34) that contains a perfect (11 of 11) match to the core consensus sequence. This exact match (bases 182 to 192) has been shown by deletion analysis to be essential for ARS function (33). A near match (10 of 11) to the core consensus sequence (bases 188 to 198) overlaps the perfect match. The 200-bp ARS307 sequence shown in Fig. 1 has been shown by deletion analysis to have optimum efficiency (33). The loss rate of a plasmid carrying this fragment (Fig. 1B) is approximately fivefold greater than that of a similar plasmid carrying ARSJ. Since plasmid stability assays, the most sensitive assays of ARS function available, can be used to measure loss rates between approximately 0.01 and 0.8 (reviewed in reference 32), mutations that increase the stability of the plasmid 5- to 10-fold or decrease the stability of the plasmid up to 3-fold could potentially be detected. For this study, we wanted to create a semisynthetic ARS element that contained as much wild-type sequence as possible and in which we could replace the core consensus sequence with synthetic oligonucleotides containing point mutations. This construct was designed to delete the overlapping 10-of-11 match to the core consensus sequence contained in ARS307 so that the effects of mutations within the perfect match could be determined without the potential complication of the redundant overlapping consensus sequence. Figure 1B shows the construction of the semisynthetic derivative of ARS307 used in this study. Palzkiil and Newlon (33) created a series of ARS307 deletion mutants that have BamHI linkers at position 1 and SphI linkers at the deletion endpoints. We identified a deletion fragment that contains wild-type sequence from residue 1 through 169 and is truncated 12 bp 5' to the T-rich strand of the core consensus sequence. When cloned into plasmid pVHA either between BamHI and SphI (plasmid pVH400) or between BamHI and Sall (plasmid pVH437), this fragment was, as expected, completely devoid of ARS activity as assayed by high frequency transformation. Using the method of Derbyshire et al. (13), which allows a single-stranded oligonucleotide to be cloned between a restriction site that has a 5' overhang and a restriction site that has a 3' overhang, we cloned a 29-base oligonucleotide into

MOL. CELL. BIOL.

plasmid pVH400 between the SphI site at the deletion endpoint and the SalI site 89 bp away in the vector, creating plasmid pVH402. This construct differs from the wild-type ARS307 sequence at two sites. It is truncated at position 195, eliminating the overlapping near match, and it contains four singlebase changes, at positions 170, 172, 173, and 176, caused by the SphI linker. The stability of the semisynthetic construct (plasmid pVH402) was found to be comparable to that of a plasmid containing a 200-bp fragment of wild-type ARS307 (plasmid pVH440). Thus, the four base changes at the SphI site and the truncation of the fragment, eliminating the overlapping near match to the consensus sequence, had no apparent effect on the efficiency of this ARS. Mutant construction and analysis. Mutations at each of the 11 positions of the core consensus sequence were made by synthesizing batches of oligonucleotides that were identical to the wild-type oligonucleotide (01) at every position except one. At this single position, an equimolar mixture of the three possible mutant bases for that position was incorporated. Eleven different batches of oligonucleotides were synthesized, each batch creating all of the possible mutations at one position of the core consensus sequence. These oligonucleotides were substituted for the wild-type oligonucleotide in M13mp8:169:01. Single-stranded phage DNAs from individual plaques were sequenced until all mutations at a given position were identified. Each mutant ARS element, which differed from the wild-type construct (M13mp8: 169:01) by one base pair, was then transferred from the phage vector to plasmid pVHA. These plasmid pVHA derivatives were tested for ARS activity and efficiency by transformation of yeast strain YNN216. To be scored as Ars+, the plasmid was required to transform at high frequency and to persist through two subcultures. The stability of all Ars+ plasmids was then determined. The fraction of plasmid-bearing cells in a culture under selection (mitotic stability) is a function of both the rate of plasmid loss and the ability of cells to continue to divide after plasmid loss (31). Experiments were performed to empirically establish the correlation between mitotic stability and rate of loss for semisynthetic ARS307 plasmids. The regression line constructed from these observations is shown in Fig. 2. This regression line was then used to calculate the rate of loss of each ARS307 plasmid from its mitotic stability. Figure 3 shows the phenotypes of the ARS core mutants. Two-thirds of the mutants were unable to transform at high frequency and to persist through at least two subcultures. These were termed Ars-. At five positions (3, 5, 8, 9, and 10), all mutations completely abolished ARS function. At all other positions of the consensus sequence, at least one mutant maintained ARS activity (Ars+). The stabilities of these mutant plasmids fell into two classes: those that were approximately equal to wild type and those with much lower stability. Only four mutant plasmids were similar to wild type in stability. Three of these plasmids carry mutations that change the ARS307 wild-type sequence to an alternate exact match to the consensus sequence (positions 1, 7, and 11). The only other mutant with wild-type activity carried an T-to-C transition at position 6. All other mutants that were able to transform at high frequency showed greatly reduced ARS efficiencies. For example, although ARS elements with all possible changes at position 1 or 11 retained function, those carrying changes to bases different from those of the consensus sequence (C and G) were less

MUTATIONAL ANALYSIS OF AN ARS CORE CONSENSUS SEQUENCE

VOL. 10, 1990

I

a

a

A

A IT IT IT JIL-

T

.198 .235 .235 .235 .020 .016 .016 .016

A

.235 t.0o16

A

n

T

9

9

G

T

_

t.016

.235

.016

9

s

T ITI T

.235 .235 .235 .235

...016 .016 ...016

.325

.303

.o.016 0-0..046 -

.i-017

.235

9 \ m \

G\

5

m

3921

.235

-----77,--;f-sX-sff-T -~ m7 - -\\\

.257 C

POS. 1

2

3

4

~~~~~~~~101 5 6 7

8

9

10 11

FIG. 3. ARS phenotypes of mutant ARS307 constructs. The plasmid pVHA/ARS307 constructs were tested for ARS activity and efficiency. The sequence of the ARS307 perfect match to the core consensus sequence is shown in the top line. The boxes below each base of the consensus sequence represent the four possible bases at each position, indicated in the left column. Thus, in each column of four bases, one box represents the semisynthetic wild-type ARS307 sequence and the other three boxes represent single-base changes at that position. Numbers inside the boxes indicate the mean and standard deviation of loss rates measured from at least three independent transformants. Symbols: [C, mnutants that were Ars- (unable to transform at high frequency); M, mutants with reduced ARS efficiency; FI1, ARS307 fragments that had wild-type efficiencies.

efficient. Similarly, mutations from T to A at positions 2, 4, and 6 resulted in dramatically lower plasmid stabilities. Two-base-pair mutation. Using the 522-bp fragment of ARS307 (34), Palzkill and Newlon created a two-base-pair mutation (522 CN6) within the perfect match to the ARS307 core consensus sequence: ATTTActTTTT (33). (The published sequence of the 522 CN6 mutation [33] is incorrect. The correct sequence of the CN6 mutation is shown here.) A plasmid carrying this mutant ARS element exhibited highfrequency transformation; however, its rate of loss was increased nearly twofold over that of a plasmid bearing the wild-type fragment. On the basis of the results shown in Fig. 4, the twobase-pair mutation in 522 CN6 might be expected to be Ars-. Singly, in the 195-bp semisynthetic ARS307 construct, the T-to-C transition at position 6 (ATTTAcGTTIT) re-

.I_ *1_ A/TI T IT wt

wt

wt

tained wild-type ARS efficiency, but the G-to-T transversion at position 7 (ATTTATtTTTT) completely abolished ARS activity. Several hypotheses can explain the Ars+ phenotype of the 522 CN6 mutant. First, in the presence of a C residue at position 6, the G-to-T transversion at position 7 might not completely abolish ARS activity. Second, the overlapping near match to the core consensus sequence (positions 188 through 198) might have core function. On the basis of the results shown in Fig. 4, this seems unlikely, since the near match (ATTTATtTTTT) was Ars- in the semisynthetic construct. Third, a combination of the overlapping near match (positions 188 through 198) and an additional near match (10 of 11 bp) to the core consensus sequence might, in combination, promote ARS activity. Using oligonucleotide-directed mutagenesis, the equiva-

.1_ T wt

I-AL-

AR

wt

wt

wt

ADNA X .

A

T

Al T IA/C T

=

wt

I1

wt

wt

X

T A/T wt

wt

MSSIO

wt

0wt k

~~~~ ~~rDNA c\\ _n22 -0,0A -0 . -0--00000000 . C,$ d ; ,;D0

POS.

1

2

3

4

5

6

7

8

9

10 11

FIG. 4. Comparison of AR5307 results with sequences of ARS elements that contain only imperfect matches (10 of 11) to the core consensus sequence. Many ARS elements contain only an imperfect match(es) to the core consensus sequence. In this figure, the positions at which the consensus sequence(s) of these ARS elements differs from the core consensus sequence is compared with the ARS307 results. The boxes in which the names of these ARS elements appear indicate the difference between the imperfect match contained in the ARS and the core consensus sequence. These boxes correspond to those shown in Fig. 3. Symbols: Fii, core consensus sequences that, in ARS307, support wild-type ARS activity; E, mutations that reduce ARS307 efficiency; = mutations that abolish ARS307 activity entirely. ARS elements shown: rDNA (rDNA ARS [38]), HO (HO ARS [23]), HMRI (HMRI ARS [1]), ARS305 (formerly ARS A6C [34]), ARS309 (formerly ARS J1lD [34]), ARS310 (formerly ARS H9G [34]), ARS137 (37), Glu3 (tRNA3 ARS [15]), and ARS3 (41). ,

u

3922

MOL. CELL. BIOL.

VAN HOUTEN AND NEWLON TABLE 1. Comparison of ARS307 fragments containing CN6 mutations

C2G1

Loss rate

Sequencea

Plasmid

200 190 GAAAACATAA ATAAAAAAGC AGTA CTTTTGTATT TATTTTTTCG TCAT

pVH402 pVH439

pVH44O

GAAAAagTAA ATAAAAAAGC AGTA

...

CTTTTtcATT TATTTTTTCG TCAG

...

190 GAAAACATAA ATAAAgtcga ccga CTTTTGTATT TATTTcagct ggct 190

(0.34)b

0.080 +0.045b

0.235 t 0.016c

...

...

CTTTTtcATT TATTTcagca ggct 200 190

...

0.323

t

0.030c

Ars-

...

0.200 t 0.006c

0.348 t 0.045c

0.560 t 0.027c

0.044 t 0.007c

...

200

190

pVH453

0.360

...

GAAAAagTAA ATAAAgtcga coga GAAAACATAA ATAAAAAAGC ggca CTTTTGTATT TATTTTTTCG ccgt

0.021b

(0.20)b

...

200

190

522 CN6

...

Mitotic stability

GAAAAagTAA ATAAAAAAGC ggca

...

CTTTTtcATT TATTTTTTCG ccgt

...

a Numbering is as in Fig. 1; all fragments begin at position 1 (left; not shown) and extend to base 522 (C2G1 and 522 CN6) or to deletion endpoints (pVH402, pVH439, pVH440, and pVH453). Uppercase letters indicate ARS307 sequence. Sequence shown includes core consensus sequence (underlined) and 5'-flanking region. Overlapping near match to consensus sequence is indicated by dots. Lowercase italics indicate CN6 mutations. Lowercase letters indicate linker or vector sequences. C2G1 and 522 CN6 were inserted at BamHI of pVHA (33). ARS307 deletion and semisynthetic fragments were inserted between BamHI and Sall of pVHA. b Data from Palzkill and Newlon (33). The loss rate was calculated by Palzkill and Newlon (33) as described in Materials and Methods but from a curve that differs slightly from that shown in Fig. 2. c Mean and standard deviation measurements of at least three independent transformants.

lent two-base-pair mutation was created in the semisynthetic ARS307 construct (plasmid pVH439; Table 1). This mutant was Ars-, demonstrating that in the presence of a C residue at position 6, the A-to-T transversion at position 7 does abolish ARS activity. This result suggests that near matches to the core consensus sequence, although individually Ars-, can cooperatively support ARS function. To test this hypothesis, the equivalent two-base-pair mutation was generated in the 200-bp ARS307 fragment from pVH440, forming plasmid pVH452 (Table 1). This plasmid was able to transform at high frequency; however, the CN6 mutation dramatically reduced the efficiency of this ARS element. These results strongly suggest that the two near matches to the core consensus sequence contained in plasmid pVH452, while each individually Ars-, can together support ARS function.

Extent of the core region. Our results demonstrate that all 11 bases of the core consensus sequence are important for ARS function. To determine whether specific nucleotides are required at positions flanking the consensus sequence, two additional experiments were undertaken. First, motivated in part by the suggestion of Kearsey (24) that a G at position 12 might be important, we synthesized oligonucleotides to determine the effect of changes at position 12 (Table 2). The wild-type ARS307 sequence has a C at position 12. Changes to T and G yielded mutant ARS elements whose efficiencies were indistinguishable from wild-type levels. However, an A at position 12 caused a twofold increase in loss rate, confirming that the base at position 12 affects ARS function. Second, we analyzed additional Bal 31 deletions that encroach on the 5' side of the core consensus sequence. ARS307 fragments that retained three or more wild-type

TABLE 2. Mutations at position 12 in semisynthetic ARS307 constructs Rate of loss' (mean ± SD)

Sequencea

Plasmid

T A

0.235

0.016

C T

A T

A T

A T

C G

A T

A

A T

A T

A T

pVH443

a t

A T

A T

A T

A T

C G

A T

T A

A T

A T

A T

T A

0.257

0.054

pVH445

c

A T

A T

A T

A T

C G

A T

T A

A T

A T

A T

T A

0.228

0.018

g t a

A T

A T

A T

A T

C G

A T

T A

A T

A T

A T

T A

0.519

0.021

pVH402(wt)

pVH444

G

A

T

12 11 10 9 8 7 6 5 4 3 2 1 POSITION a Sequence corresponds to bases 181 to 192 in Fig. 1. Lowercase letters indicate change from wild-type sequence. The match to the 11-bp core consensus sequence is underlined. I Measured from at least three independent transformants.

VOL. 10, 1990

Plasmid

pVH44O

MUTATIONAL ANALYSIS OF AN ARS CORE CONSENSUS SEQUENCE TABLE 3. Effect of deletions 5' to ARS307 core consensus sequence Sequencea 190 200 ... GAAAACATAA ATAAAAAAGC ggcatg ... CTTTTGTATT TATTTTTTCG ccgtac 190

pVH442

pVH4O2 pVH441

Rate of lossb (mean + SD)

0.200

0.006

198

... GAAAACATAA ATAAAAAAgg catg ... CTTTTGTATT TATTTTTTcc gtac 190 ... GAAAACATAA ... CTTTTGTATT 190 ... GAAAACATAA ... CTTTTGTATT

3923

ATAAAgtcac c

0.219 ± 0.026

0.235 ± 0.016

TATTTcagtg g ATggcatgca TAccgtacgt

0.450 ± 0.060

a Numbering as in Fig. 1. Lowercase letters indicate SphI or SailI sequences. The perfect match to the consensus sequence is underlined. Near match to the core consensus sequence marked by dots. All constructs were cloned between the BamHI and SalI sites of pVHA. b Measurements of at least three independent transformants.

residues 5' to the T-rich strand of the core consensus sequence retained approximately wild-type efficiency (plasmids pVH440, pVH442, and pVH402; Table 3). Deletion of these three bases (plasmid pVH441) reduced ARS efficiency approximately twofold. These results suggest that bases immediately 5' to the core consensus sequence are also important for ARS activity. DISCUSSION The ARS core has been defined as a region that is essential for ARS function. The consensus sequence, first suggested by Stinchcomb et al. (41) and later extended by Broach et al. (8), occurs within the core region. It has been difficult to refine this consensus sequence because most ARS elements contain multiple matches to the sequence and in only a few cases has a single match been shown to be essential for function. In this study, we have investigated the importance to ARS307 activity of each of the 11 positions of the core consensus sequence proposed by Broach et al. (8). Our results demonstrate that in ARS307, mutants that bear alternate perfect matches to the core consensus sequence but differ from that contained by ARS307 (positions, 1, 7, and 11) have efficiencies approximately equal to wild-type level. In addition, these experiments have shown that a mutant with a C in position 6 also retains wild-type activity. No yeast chromosomal ARS element that bears a single core consensus sequence with C at position 6 has yet been sequenced. ARS310 contains three imperfect matches to the core consensus sequence (34). One small DdeI subclone of ARS310 that is Ars+ includes only the near match with a C at position 6. The core consensus sequences of several ARS elements isolated from other organisms (heterologous ARS elements) also have a C at position 6. Of the heterologous ARS elements that contain only a single near-perfect match to the core consensus sequence, those with a C at position 6 include the Drosophila mitochondrial fragment HHa 240 (29), the M13 mutant fragment D10 (25), and the Chlamydomonas chloroplast fragment 01 (47). In addition, the pSRI plasmid of Zygosaccharomyces rouxii contains an ARS element with two near-perfect matches. Of these, the near match that contains a C at position 6 has been shown by deletions and point mutations to be essential for function (2). Therefore, in the context of several different flanking sequences, a core consensus sequence with a C at position 6 is able to provide ARS function.

All other mutations within the 11-bp region of ARS307 either dramatically reduce or completely abolish ARS function. This finding demonstrates that each position within the 11-bp consensus sequence is important for the function of ARS307. We have also investigated the DNA sequence requirements at the position flanking the 3' side of the core consensus sequence (position 12) and find that it also contributes to ARS307 efficiency. C, G, and T residues in this position result in wild-type efficiency, whereas an A at position 12 causes a twofold reduction in plasmid stability. These results confirm and extend the experimental findings of Kipling and Kearsey (25) as well as the sequence analysis of Kearsey (24). The experiments of Kearsey (24), Bouton and Smith (4), Strich et al. (44), and Araki and Oshima (2) indicate that bases 3' to position 12 may also be critical to ARS function. On the basis of our findings, we propose a modified and expanded ARS core consensus sequence: 5' - T T T T A T A T T T T T - 3' A C G A C G

The DNA sequence requirements 5' to the T-rich strand of the core consensus sequence are less clear. The results of the experiments reported here, the sequence comparisons of Sinha et al. (37), as well as deletion mutations created in ARS1 (44), the HO ARS (24), and the H4 ARS (4) indicate that bases immediately 5' to the core consensus sequence are also very important for ARS function. However, a linker scanning substitution mutation immediately adjacent to the 5' T of the H4 ARS core consensus sequence did not appear to affect ARS function (4). Some ARS elements do not contain an exact match to the consensus sequence but instead contain one or more 10of-11 matches. A comparison of these near matches to the results of the ARS307 mutagenesis results is shown in Fig. 4. This comparison reveals a paradox because several of the ARS elements, including ARS3, the tRNA lu ARS, ARS305, and ARS309 contain only near matches that are nonfunctional in the ARS307 context. In the case of ARS elements that bear multiple near matches to the core consensus sequence (ribosomal DNA [rDNA] ARS, ARS305, ARS309, and ARS310), only a few of the near matches are Ars+ in the semisynthetic ARS307 constructs. The rDNA ARS and ARS310 each contain one near match with a C at position 6, a mutation that, in ARS307, retains wild-type efficiency. The

3924

VAN HOUTEN AND NEWLON

rDNA ARS also contains one near match with a G in position 1, a mutation that reduces ARS efficiency in ARS307. There are several possible explanations for this paradox. First, ARS307 is less efficient than some ARS elements; for example, plasmids carrying ARS307 are lost at a rate five- to sixfold higher than are ARSI-containing plasmids. It is possible that some sequence or DNA conformation that contributes to ARS efficiency is missing from ARS307. In the presence of such an element, some of the mutations that abolished ARS307 activity might retain function. The consensus sequence in the tRNA Glu ARS (TTTTATATgTT) is an example of such a sequence. Second, in the case of yeast ARS elements with multiple 10-of-11 matches, it has not yet been determined whether a single near match is sufficient for function. It is possible that in this type of ARS, each 10-of-11 match contributes to ARS activity. If, for example, the core is a replication initiation protein-binding site, then each of the near matches may bind that protein at a low efficiency. The combined low efficiencies of all of the near matches may be sufficient for detectable ARS activity. This cooperative contribution of several near matches to the core consensus sequence has been observed in several heterologous ARS elements. Kipling and Kearsey (25) generated several heterologous ARS elements by mutating M13 sequences. The mutant Ars+ fragments differed from the Ars- wild-type sequences at positions that created additional close matches to the core consensus sequence. Zweifel and Fangman (48) studied a fragment of yeast mitochondrial DNA that contained multiple close matches to the consensus sequence but that lacked ARS activity when present on plasmids in a single copy. By placing multiple copies of this fragment in YIp5, a plasmid capable of high-frequency transformation was produced. The two-base-pair CN6 mutations provide further evidence in support of this hypothesis. In the absence of other near matches to the core consensus sequence, the CN6 mutation (ATTTACtlTT.TT, a 10-of-11 match by the proposed consensus sequence) in the semisynthetic ARS307 construct (plasmid pVH439) was unable to sustain ARS activity. In the presence of a second Ars- near match to the consensus sequence (ATTTATITl-l7, plasmid pVH452), weak ARS activity was observed. These data suggest that multiple near matches to the core consensus sequence each contribute to ARS activity. The core of an ARS element has been defined as a region essential for function. By analogy to the simian virus 40 origin of replication (see references 10 and 40 for reviews), the ARS core would be both necessary and sufficient for ARS activity. Subcloning experiments performed by Srienc et al. (39) have shown that in at least some plasmid contexts, the minimal ARSI fragment is 15 bp long and includes the core consensus sequence. On the basis of deletion analysis, Kearsey (24) estimated the size of the smallest HO ARS fragment to be a 14-bp fragment that includes the core consensus sequence. However, the 14-bp HO ARS fragment has not been tested for function. Deletion analyses of ARS307, the histone H4 ARS, and the HMR E ARS indicate that the minimal fragments of these ARS elements that retain function are much larger, in the range of approximately 50 to 100 bp (4, 6, 18, 33). The differences in the estimates for the size of the smallest ARS fragment sufficient for function may result in part from differences in vector context and procedural differences. However, there may also be inherent differences between ARS elements. The experiments presented here demonstrate that in ARS307, each base of the

MOL. CELL. BIOL.

core consensus sequence, as well as bases immediately flanking both sides of the consensus sequence, contributes to ARS function. Experiments that will further define the extent of the core region of ARS307 are under way. ACKNOWLEDGMENTS We thank Marjorie Brandriss, Walton Fangman, Mike Newlon, Jim Theis, Scott Greenfeder, and Irene Collins for their valuable comments on the manuscript. This work was supported by Public Health Service grant GM35679 from the National Institutes of Health. LITERATURE CITED 1. Abraham, J., K. A. Nasmyth, J. N. Strathern, A. J. S. Klar, and J. B. Hicks. 1984. Regulation of mating-type information in yeast: negative control requiring sequences both 5' and 3' to the regulated region. J. Mol. Biol. 176:307-331. 2. Araki, H., and Y. Oshima. 1989. An autonomously replicating sequence of pRSI plasmid is effective in two yeast species, Zygosaccharomyces rouxii and Saccharomyces cerevisiae. J. Mol. Biol. 207:757-769. 3. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant DNA. Nucleic Acids Res. 7:1513-1523. 4. Bouton, A. H., and M. M. Smith. 1986. Fine-structure analysis of the DNA sequence requirements for autonomous replication of Saccharomyces cerevisiae plasmids. Mol. Cell. Biol. 6: 2354-2363. 5. Bouton, A. H., V. B. Stirling, and M. M. Smith. 1987. Analysis of DNA sequences homologous with the ARS core consensus in Saccharomyces cerevisiae. Yeast 3:107-115. 6. Brand, A. H., G. Micklem, and K. Nasmyth. 1987. A yeast silencer contains sequences that can promote autonomous plasmid replication and transcriptional activation. Cell 51:709-719. 7. Brewer, B. J., and W. L. Fangman. 1987. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell

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