Current Genetics

Curt Genet (1992)22:175-180

9 Springer-Verlag 1992

Original articles Mutational analysis of a variant of ARS1 from Saccharomyces cerevisiae Finn Kirpekar and Kay Gullov Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark Received January 20, 1992

Summary. A naturally occurring single base-pair G to A transition, creating a 10/11 near-match close to the essential 11 base-pair core consensus of ARSI, was used to investigate the importance of near-match sequences. The 10/11 near-match can not substitute for the core consensus since an ARS- phenotype is observed when the core consensus is deleted. However, deletion mutations revealed that this near-match together with a short palindromic sequence, also situated in the B-flanking region, comprise a single element crucial for optimal ARS function. The palindrome has the potential of forming a stemloop structure. Rather precise observations concerning the borders of the B-region were achieved. The four base pairs separating the near-match from the core consensus perform a spacing function where the identity of the bases are unimportant. However, this spacing is highly important since deletion of these four base pairs leads to an ARS- phenotype. Key words: ARS1 mutants - DNA replication - Yeast Single-stranded DNA

Introduction The autonomously replicating sequence ARS1 from the yeast Saccharomyces cerevisiae can be isolated on a 1.45 kb EcoRI fragment also habouring the gene, TRP1, for phosphoribosyl-anthranilate isomerase (Stinchcomb et al. 1979; Tschumper and Carbon 1980). ARS1 confers on plasmids the ability to replicate autonomously in yeast cells and is thus a likely candidate for a chromosomal origin of replication. ARS1 has been shown to serve as an origin or replication in plasmid contexts (Brewer and Fangman 1987) although it has not been proven to be a true origin of replication in the chromosome. Comparison among different ARSes has shown the J 1 base-pair (bp) core consensus 5'-(A/T)AAA(C/ Correspondence to: E Kirpekar

T)ATAAA(A/T)-3' to be the only common element (Broach et al. 1983). These 11 bp are necessary, but not sufficient, for ARS function. The core consensus is situated in an AT-rich region which may be important for the formation of single-stranded DNA regions during initiation of replication. This is supported by experiments showing that a DNA unwinding element (DUE), rich in A-T base pairs, is essential for the function of the H4 ARS (Umek and Kowalski 1988). In addition to the 1t bp core consensus all sequenced ARSes carry several near-matches (9 or 10 out of 11) to the core consensus. The near-matches and their orientation relative to the core consensus appear to be important for the function of ARS307 (Palzkill and Newlon 1988). In contrast, nearmatch sequences do not play a significant role in the function of the H4 ARS (Holmes and Smith 1989). The sequences flanking the core consensus are of importance for ARS1, but successive deletions have not revealed any clear boundaries for ARS1. The ARS efficiency is reduced with increased deletions leaving as little as 19 bp including the 11 bp core consensus as a functional, but very inefficient, ARS fragment (Srienc et al. 1985). We have isolated a naturally occurring ARSI with a point mutation creating a 10/11 match separated by just 4 bp from the core consensus. The significance of this nearmatch and an interrupted palindrome in the same region, the B domain of ARS1, was investigated by deletion mutations.

Materials and methods Strains, media and growth conditions. The S. cerevisiae strain used, DBY746 (Matc~, his3, leu2, ura3, trpl), was grown at 30 ~ either in Wickerham medium supplemented with histidine, uracil, leucine and glucose, to maintain plasmids, or.in YEPD medium when not containing plasmid. The E. coli strain HB101 was used for cloning and purification of plasmid DNA. DNA isolation and transformation. E. coli plasmid DNA was isolated from cells grown in L-Broth with 50 btg/ml of ampicillin by the alkaline lysis method as described by Sambrook et al. (1989). Bulk

176 yeast DNA was prepared according to Winston eta1. (1983). DBY746 was transformed as described in Ito et al. (1983). DNA cloning and sequencing. Isolation of the TRP1-ARS1 fragment from Y379-5D has been described previously (Kielland-Brandt et al. 1981). pSA1452 was made by cloning the 1.45 kb TRPI-ARS1 fragment into the EeoRI site of pBR322 (Gullov and Friis 1985). pASBI and pASB2 were constructed by cutting pSAI452 at the unique sites for StuI and BglII. The BglII site was made blunt-ended either by filling out with the Klenow fragment from DNA polymerase I and all four dNTPs (pASBI) or by removing the sticky ends with mung bean nuclease (pASB2). The plasmids were subsequently circularized with T4 DNA ligase, pAARS6 was made by treating BglII-digested pSA1452 with T4 DNA polymerase (1 unit/ gg DNA) making use of the potent 3'-5' exonuclease enzyme activity; 100 gM of dATP was added to limit the extent of the deletion to the first A nucleotide encountered by the enzyme. The DNA was made blunt-ended with mung bean nuclease (MBN) and circularized with T4 DNA ligase, pABI and pAB7 were constructed by digesting pSA1452 with BglII, making blunt ends with MBN and circularizing with T4 DNA ligase. To construct pAP, pSAI452 was first digested with BamHI and SspI and the two fragments (3.8 kb and 2.0 kb) were separated by agarose-gel electrophoreses and subsequently purified. The 3.8 kb fragment was methylated at the PstI site in the /3-1actamase gene and rejoined to the 2.0 kb fragment using T4 DNA ligase. The construct was then digested with PstI, the 3' overhang removed with MBN and the DNA re-circularized. pASF was made by using a FspI site at position 820 in pASS2. An 0.57 kb FspI-DraIII fragment from pASS2, harbouring ARS1, was ligated into the 5.2 kb StuI-DraIII fragment from pSA1452 resulting in the deletion of position 830-840. pAN was constructed by replacing the 22 bp StuI-BglII fragment in pSA1452 with a synthetic fragment not containing the near-match at position 842-852. All mutants were sequenced except for pASF whose identity was confirmed by the presence of a BglI site created by the deletion. To sequence the ARS! region, the 838 bp HindIII-EcoRI fragment containing A RS1 and flanking regions was cloned into pUC 19 and sequenced using the dideoxy nucleotide method according to the protocol supplied with the SequenaseTM enzyme (United States Biochemical Co. Cleveland, OH). Growth parameters and plasmid copy number. Yeast culture doubling-time was determined by measuring the optical density at 500 nm. The mitotic stability was measured by plating from exponentially growing cultures (OD55o =0.5) in minimal medium onto SD minimal plates (supplemented with uracil, histidine and leucine) with and without tryptophan, respectively. To register the plasmid loss rate, exponentially growing cultures in minimal medium at ODsso=0.2 was supplemented with 20 gg/ml of tryptophan and aliquots were taken every second hour to follow growth and the number of plasmid-containing cells. The relative number of plasmids in yeast cultures was estimated from slot blots: serial dilutions of total DNA from the same number of cells harbouring the wildtype plasmid and mutant plasmids were blotted onto a Zeta-Probe nylon supported membrane on a Bio-Rad (Richmond, CA) slot blot apparatus according to the manufacturers, instructions. The filters were hybridized with e_32p random primer extension-labeled (Feinberg and Vogelstein 1983) pBR322 and exposed on a Kodak Exomat AR film. The autoradiograms were scanned densitometrically on an LKB ultroscan XL. To correct for varying yields during DNA preparation the slot blot filters were re-hybridized with a 0.66 kb rDNA probe (Petes et al. 1978). MBN relaxation ofplasmids. Was performed as described (Sheflin and Kowalski 1985) using the restriction enzymes EcoRI, StyI and XbaI to localize the MBN-sensitive regions. To map the MBN incision sites at the nueleotide level, t0 gg of MBN-relaxed plasmid was digested with HindIlI and NheI and the 422 bp fragment containing ARS1 was purified from an agarose gel. The fragment was end-labeled at either the HindIII site or the NheI site using e.3zp. dATP and ct-32P-dCTP, respectively. The end-labeled fragments

were electrophoresed through a 4% sequencing polyacrylamide gel alongside purine sequencing reactions (Ambrose and Pless 1987) on identical fragments from non-relaxed plasmids.

Results Effect o f deletions in the B-domain As shown in Fig. 1 A, sequencing of the region around the ARS core consensus on a 1.45 kb T R P 1 - A R S 1 fragment isolated from the yeast strain Y379-5D revealed a G to A transition at position 845 compared to the previously published sequence (Tschumper and Carbon 1980). This creates a 10/11 match (5'-CAAACATAAAA-Y, position 842-852) in the same orientation as, and just next to, the core consensus (5'-TAAACATAAAA-Y, position 857-867). This polymorphism was the only change observed in the 355 bp sequenced region (position 6 1 5 970). The T R P 1 - A R S 1 fragment has previously been cloned into the E c o R I site of pBR322, generating the plasmid pSA1452 (Gullov and Friis 1985) which was used for the construction of deletion mutants and for transformation into yeast. Figure 1B shows the the abundance of 9/11 and 10/11 near-matches in the part of the T R P I - A R S 1 fragment we have sequenced. Several others are located outside this fragment (Palzkill and Newlon 1988). Actually, all ARSes sequenced have a frequency of 9/11 and 10/11 nearmatches, 8- to 30-times higher than what would be expected from the base composition implying that the nearmatches are significant for ARS function. Indeed, in the somewhat inefficient A R S 3 0 7 it has already been demonstrated that the presence and orientation of the nearmatches are of importance (Palzkill and Newlon 1988). Considering these observations, it is tempting to speculate that the nature of our mutation, changing a 9/11 near-match to a 10/11 near-match, is not a mere coincidence. To clarify whether the 10/11 near-match has a significance for the function of A R S 1 , we constructed the plasmid pAN deleting the near-match at position 842-852. Transformed into the yeast strain DBY746, pAN gave rise to a culture with a doubling time of 3.5 h and 6.5% of plasmid-containing cells in medium without tryptophan; the culture lost the plasmid at a rate of 24% per generation after tryptophan was added. As estimated by hybridization, the average number of plasmids per cell was only 20% of that in a wild-type plasmid-harbouring culture. This clearly demonstrates that the deletion, removing the near-match, affects the replication of the plasmid. However, the mutants pASF, pASS2 and pASS3, removing 12, 18 and 22 base pairs respectively just upstream of the 10/11 near-match (Fig. 2), have approximately the same growth parameters as pAN (Table 1) showing that the near-match is not the only part of the B-region important for the function of A R S 1 . It is noteworthy that the three deletion mutants, pASF, pASS2 and pASS3, eliminate a 10 bp interrupted palindromic sequence at position 831-841. The mutants pASB1 and pASB2 (Fig. 2), which lack both the 10/11 near-match

177 Table 1. Effect of deletions within the B-domain of A R S I

I 600

9 m

~ 700

9 --

9 m 800

10 11 ----

I 900

10 --

I 1000

S l. A Sequence of the variant A R S I around the core consensus. Sequencing was performed by the dideoxy termination method and the reactions electrophoresed through a 6% sequencing polyacrylamide gel. The arrow shows the position of the 845 guanine to adenine transition. Numbering here and in the following figures are according to Tschumper and Carbon (1980). B Abundance of nearmatches around A R S I with the number of bases homologous to the 11 bp core consensus. Boxes above the line designate near-matches with their th3,mine-rich strands following the numbering; boxes below the line designate near-matches with their adenine-rich strands following the numbering Fig.

ASS2

i

AB7

ASS2 ~

ABI ~SBI ~ ASB2 ~

iG

ASF ~

I

G CA CT TG C CTG CAGG CC TTTTGAAAAG

a{s

82o

8~o

~

&ARS6

I--

io CAAACATAAAAGATC

I

ii TAAACATI%AAAT CT

"

Fig. 2. Map of the deletion mutants constructed in this work. Underlined sequences, marked 10 and 11, designate the 10/11 nearmatch and the core consensus respectively. Arrows show the major MBN incision sites with the arrow above the sequence indicating an incision in the thymine-rich strand relative to the core consensus and the arrows below the sequence indicate incision in the opposite strand. The G at position 855 in ASB1 refers to a point mutation generated during cloning

and the interrupted palindrome, affect A R S 1 replication to nearly the same extent as the mutants removing just one of the two elements, suggesting that the palindrome and the near-match together comprise a single element necessary for optimal A R S 1 function. The left border of this element is delimited by our mutations: since there is little difference in growth between pASS2-, pASS3- and pASF-harbouring cells, the sequence 819-829 is probably dispensable for A R S function. This notion is supported by the deletion m u t a n t pAP, lacking position 8 2 3 827, which exhibits the same growth rate, plasrnid stability and average copy n u m b e r as the wild-type. Thus, it appears that the left border of the element 5' to the core consensus extends no further than position 828. H o w ever, it should be stressed that others have reported sequences further upstream to be i m p o r t a n t for A R S 1

Mutant plasmid

Doubling time (h)

Milotic stability ~

Loss rate

Copy no. per cellb

Wild-type pASS2 pASS3 pAP pASF pAN pASB1 pASB2 pAB1 pAB7 pAARS6

3.3 3.7 3.8 3.5 4.2 3.5 4.5 3.8 4.2 -

14__+2.5 4.4 +0.5 4.3 + 0.5 12_+1.5 5.3 • 0.5 6.5 • 1.0 6.54-1.5 6.7+ 1.5 2.8__+0.5 < 0.01 < 0.05

16 25 25 18 26 24 25 28 40 -

100 20 +_5 20 • 5 100• 20 • 5 20 • 5 10 • 10+__3 5• -

a Mitotic stabilities are mean values of triplicate estimations b Percent of wild-type

(Snyder et al. 1986; Diffley and Stillman 1988), observations not in conflict with our results. Our experiments can not rigorously determine whether the 5'-flanking element is consecutive with the core consensus. F r o m the construct pASB2, it is evident that the 4 bp separating the near-match from the core consensus are not essential for the function of A R S I . However, a deletion mutant removing only these four base pairs (pAB7; Fig. 2) gave rise to a high frequency of abortive transformants which could occasionally be restreaked but were unable to grow in liquid medium without tryptophan. The fraction of plasmid-containing cells in colonies was less than 1 in 104. This apparent p a r a d o x m a y be explained by the requirement for a certain spacing between the core consensus and the I0/11 near-match where a juxtaposition of these two elements renders A R S ! non-functional. This hypothesis is supported by the f a c t that pAB1, lacking one base pair more than pAB7, generates viable transformants (Table 1) implying that the significance of the four bases, separating the near-match from the core consensus, does not reside in the primary sequence. Recently, a protein factor (ACBP), binding to the thymine-rich strand of the single-stranded core consensus, has been identified ( H o f m a n n and Gasser 1991). It was reported that this protein also bound near-matches, suggesting a model for initiation of replication where the core consensus and a near-match in the opposite orientation should constitute the starting points for leadingstrand D N A synthesis. As seen in Fig. 1 B, a 9/11 nearmatch in opposite orientation is present around position 800. In order to investigate if this 9/11 near-match together with the 10/11 near-match could constitute a functional ARS, the plasmid pAARS6, deleting position 8 5 3 - 863 (Fig. 2) which includes the first seven bases of the core consensus, was constructed, pAARS6 gave rise to a high frequency of abortive transformants with the same phenotype as pAB7. This clearly demonstrates that the 10/11 near-match, although required for optimal A R S 1 function, can not substitute for the core consensus thus confirming the need for adenine or thymine at the first position in the core consensus.

178 Nhel

EcoRI 0/5816 Xbol

/

Hi'rid lit

5588

/ /

189

SlyI

[

4447 "--~

~ Mojor MBN . . _ x ~ . ~ incision region

5788 ....

/

r .....

.,

..........

_cA

Hindl~

\opp.85o V"

p O ~ I~O~-

-~

Nh~

\ 1037

4tc'~

L_. EcoRI

"t MinorMBN incision region app. 2450

Fig. 3. Map of pSA1452 with the restriction enzyme sites used to locate the MBN-sensitive regions. Amp denotes the fl-lactamase gene in pBR322

Fig. 4. MBN incision pattern mapped at the nucleotide level. From MBN-relaxed pSA1452, the 422 bp HindIII-NheI fragment harbouring ARSI was purified and 32p end-labeled at either end. The fragments were electrophoresed through a 4% sequencing polyacrylamide gel. Lane I, purine sequencing reaction on the thymine-rich strand (relative to the core consensus); lane 2, MBN incision on the thymine-rich strand; lane 3, MBN incisions on the adenine-rich strand; lane 4, purine sequencing reaction on the adenine-rich strand

Single-stranded regions in A R S 1

The secondary structure of the D N A in pSAI452 and mutant plasmids was investigated using the single-strandspecific enzyme MBN. The underwinding o f the D N A double helix in plasmids is generally thought to result in negative supercoiling of the plasmid; alternatively the underwound state may also result in partial unwinding of the double helix or the adoption of other secondary structures. Since generation of M B N sensitivity is dependent on the plasmid being underwound, which was confirmed

Fig. 5. MBN incision pattern in wild-type and mutant plasmids: MBN-relaxed plasmids were digested with EcoRI, 32p end-labeled and denaturated by glyoxylation prior to electrophoresis through a 1% agarose gel. Lane 1, pSA1452; lane 2, pASS2; lane 3, pASS3; lane4, p6SB1; lane5, pASF; lane6, pAN; lane 7, marker. Open arrows show the bands generated due to incisions in the terminator region of the/~-lactamase gene; closed arrows show the bands generated from the incisions around ARSI

on topoisomerase I-relaxed, closed, plasmids (data not shown), only a single nick, allowing the plasmid to become relaxed, will be made in each plasmid by M B N thus making it possible to compare the sensitivity at different sites. In short, supercoiled plasmids isolated from E. coil and dissolved in 10 m M Tris, p H 7.0, were incubated with M B N to allow relaxation of the D N A and were subsequently cut with a restriction enzyme and end-labeled. Following electrophoresis through a denaturating agarose gel, the fragments were blotted onto a nitrocellulose filter and autoradiography was performed (an example using E c o R I is shown in Fig. 5). In pSAI452, two MBN-sensitive regions are found as summarized in Fig. 3: one is located at the end of the 3-1actamase gene as previously reported (Sheflin and Kowalski 1985); the other is found around A R S 1 which parallels the situations in the H4 ARS (Umek and Kowalski 1988) and the 2 gm ARS (Umek and Kowalski 1987) where MBN-sensitive regions are also found. The M B N incision pattern in A R S 1 was also mapped at the nucleotide level. Figure 4 shows that the incisions are found in the core consensus and in both its flanking regions. The three most prominent bands are 5' to the core consensus, two at position 836 and 837 on the adenine-rich strand and one at position 820 on the thymine-rich strand. The major M B N incisions on the adenine-rich strand are on both sides of a G situated in the middle of the 10 bp interrupted palindromic sequence which is important for the function of A R S 1 . This palindrome probably forms a stem-loop structure which makes the phosphodiester bonds on both sides of the G at position 836 particulary sensitive to MBN. F o r unknown reasons the thymine-rich strand does not display a similar MBN-sensitive structure at position 836. It should be stressed that the M B N patterns on A R S 1 shown in Fig. 4 are not directly comparable to the previously published patterns in the H4 ARS and the 2 gm ARS (Umek and Kowalski 1987, 1988), since these con-

179 tained 1 mM of EDTA during the MBN reaction which affects the DNA conformation by chelating divalent cations. Performing the MBN assay on A R S I with EDTA, we observe a much more dispersed incision pattern covering the region 740 to 920 (data not shown). Thus, our results show that a region in the B-domain of ARS1, clearly important for ARS function, has the potential of forming a pronounced secondary structure adjacent to the core consensus given the right ionic conditions. We also investigated the effect of removing the most MBN-sensitive sites on the unwinding-ability of A RS!. A series of ARS1 mutants were relaxed using MBN and digested with EcoRI prior to electrophoresis (Fig. 5). The relative sensitivity for MBN in the A R S I region can be estimated by comparing the fraction of nicking in the /~-lactamase terminator region to the fraction of nicking in the ARS1 region. The wild-type plasmid and all the mutants tested have essentially the same MBN sensitivity around A R S I . Neither the removal of the palindrome (pASF), the near-match (pAN), or both (pASB1), affect the unwinding capacity of ARS1. The same is true for the mutant plasmids pASS2 and pASS3 lacking either the two MBN-sensitive sites in the palindrome or all three MBNsensitive sites, respectively. From this we conclude that the function of the B-domain, adjacent to the core consensus, is not to promote a general DNA unwinding. Rather, we believe that the element covering the palindrome and the 10/11 near-match is recognized by factors participating in the replication machinery, most likely through the adoption of a stem-loop structure.

Discussion

We have isolated a variant of A R S ! from S. cerevisiae with a G to A transition at position 845 which changes the 9/11 near-match just next to the core consensus into a 10/11 near-match. Taking into consideration the high frequency of near-matches in all ARSes, it appeared to us that the nature of this change might be more than a coincidence. From the phenotype of our deletion mutants it can be seen that the 10/11 near-match together with a 10 bp interrupted palindrome, 5' to the near-match, comprise a single element which must carry out a sort of auxilliary function to ensure optimal ARS activity. The maximum size of this element is 25 bp (position 828 to 852). The interrupted palindrome may, under certain conditions, form a stem-loop structure, but the presence of the palindrome is not a prerequisite for the general unwinding ability of ARS1. Deleting most of the core consensus generates an ARS- phenotype thus showing that the 10/11 nearmatch, in an otherwise conserved context, does not constitute a functional ARS. This is in apparent contrast to ARS307 where a mutation to a C at the first position in the core consensus reduces, but in no way abolishes, ARS efficiency (Van Houten and Newlon 1990). A likely explanation is that the 10/11 near-match in A R S I , being part of an auxiliary element, is unable, at the same time or at that particular position, to substitute for the core consensus. In the mutated ARS307 the region corre-

sponding to the B-region in ARS1, with its auxilliary functions, is intact. The four bases separating the 10/11 near-match from the core consensus perform an essential spacing function where the identity of the bases is unimportant. It is evident that the lack of a certain spacing or phasing between the 10/11 near-match and the core consensus is detrimental. The properties of the recently identified protein ACBP (Hofmann and Gasser 1991), which binds certain near-matches as well as the core consensus, may account for this. A juxtaposition of these two elements may cause ACBP to interact with them in a way which prevents replication. Alternatively, it is possible that the 10/11 near-match simulates some of the functions performed by the core consensus and, in a competitive manner, could interfere with replication when the two elements are fused. In conflict with our results, it has previously been reported that ARS1 is not affected by removal of the 4 bp between the core consensus and the near-match (Diffiey and Stillman 1988). The version of ARS1 used in that work does not have the position 845 G to A transition in the near-match, which implies that an improved homology of a near-match to the core consensus, in certain contexts, has a negative effect on the ARS. The present version of ARS1 has not revealed any characteristics different from the previously identified version (Tschumper and Carbon 1980): cloned into pBR322, the TRPI-ARS1 fragment confers upon the plasmid a mitotic stability and loss rate which is comparable to that of other reports (Stinchcomb et al. 1979). When propagating the TR P 1-A R S! fragment as an endogeneous plasmid, we observe a high mitotic stability and very low loss rate (data not shown) in agreement with earlier reports (Zakian and Scott 1982). Hence, it seems that the position 845 G to A transition does not have any effect on the efficiency of ARS1. We can only speculate on the function of the many near-matches since very little is known about the initiation of DNA replication in eukaryotes. One function could be the proposed binding of ACBP to a near-match in opposite orientation to the core consensus thus forming a second starting point for leading-strand DNA synthesis. However, this can only explain the presence of a single near-match. Another function could be that the near-matches are storage-sites for factors needed during initiation of replication making them easily accessible. These two hypothesis are not mutually exclusive. We have shown that ARS1 contains a DUE when isolated from E. coli in a plasmid context. The unwinding depends on the DNA being under torsional stress. There are conflicting results concerning DNA being under unconstrained torsional stress in chromatin. Bulk 2 gm minichromosomes from yeast do not contain DNA under torsional stress (Saavedra and Huberman 1986), while both positive (Ambrose et al. 1987; Barsoum and Berg 1985) and negative (Petryniak and Lutter 1987) results are reported regarding unconstrained torsional stress in SV40 chromatin. Though DNA in chromatin may not generally be under torsional stress, and thus does not spontanously unwind, this does not imply that the DUE

180 is w i t h o u t i m p o r t a n c e for ARS1 function. First, unwinding o f the D N A at an origin o f replication is still required, no matter h o w this is accomplished, and a low energy o f unwinding will be beneficial. Second, it is also possible that a cell cycle-dependent subfraction o f the D N A is u n d e r torsional stress m a k i n g the D N A u n w o u n d at the a p p r o p r i a t e time within the S-phase. Indeed, the H 4 A R S exhibits a cell cycle-dependent D N A s e I sensitivity pattern as an indication of a d y n a m i c D N A c o n f o r m a t i o n (Brown et al. 1991).

Acknowledgements. The authors thank S. Andersen for technical assistance. This work was supported by a yeast career grant from Nordic Yeast Research program, grant no. D-1038-101, to E Kirpekar.

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C o m m u n i c a t e d by B. S. Cox