268 Biochimica et Biophvsica Acta. 951 (1988) 268 273 Elsevier

BBA 91887

T h e yeas t D N A po lymerase -pr imase complex: genes and prote ins

P. P levan i , M. F o i a n i , M. M u z i F a l c o n i , A. P izzagal l i , C. S a n t o c a n a l e ,

S. F r a n c e s c o n i , P. Va l sa sn in i , A. C o m e d i n i , S. P ia t t i a n d G. L u c c h i n i

Dipartimento di Genetica e di Biologia dei Mieroorgansimi, Universith degli Studi di Milano, Milan (Italy)

(Received 21 September 1988)

Key words: DNA replication; DNA polymerase-primase; Gene structure; (Yeast)

The yeast DNA polymerase-primase complex is composed of four polypeptides designated plS0, p74, p58 and p48. All the genes coding for these polypeptides have now been cloned. By protein sequence comparison we found that yeast DNA polymerase I (a) shares three major regions of homology with several DNA polymerases. A fourth region, called region P, is conserved in yeast and human DNA polymerase a. The site of a temperature-sensitive mutation in the P O L l gene which causes decreased stability of the polymerase- primase complex has been sequenced and falls in this region. We hypothesize that region P is important for protein-protein interactions. Highly selective biochemical methods might be similarly important to dis- tingnish functional domains in the polymerase-primase complex. An autocatalytic affinity labeling procedure has been applied to map the active center of yeast DNA primase. From this approach we conclude that both primase subunits (p48 and p58) participate in the formation of the catalytic site of the enzyme.

Introduction

A eukaryotic DNA polymerase activity was first discovered in calf thymus and successively named DNA polymerase a [1]. Later, three more DNA polymerases were found in eukaryotic cells and named fl, T and 8 [2]. DNA polymerase T is the mitochondrial enzyme and DNA polymerase fl is generally regarded as the polymerase activity involved in DNA repair. Various experimental evidence indicates DNA polymerase a as the major replicative enzyme. However, recent studies sug- gest that DNA polymerases a and 8 might be both involved in the replication, respectively, of the lagging and leading strand of the replication forks [3]. It would be advantageous for the lag-

Correspondence: P. Plevani, Dipartimento di Genetica e di Biologia dei Microrganismi, Via Celoria, 26, 20133 Milano, Italy.

ging-strand DNA replicase to be associated with a DNA primase activity to catalyze the formation of RNA primers necessary for the de novo synthesis of the Okazaki fragments [4]. Indeed, a DNA primase has been found tightly associated with DNA polymerase a in a variety of eukaryotic organisms [2]. The lack of DNA polymerase mutants and the difficulties in applying powerful genetic methods to higher eukaryotic cells can be partially overcome by using the yeast Sac- charomyces cerevisiae as a model system to study the molecular mechanisms of DNA replication.

Initial biochemical analysis in this simple eukaryote identified a mitochondrial DNA poly- merase [5] and two high-molecular-weight nuclear DNA polymerases, named DNA polymerases I and II [6]. Recently, a third yeast enzyme, with biochemical properties similar to DNA poly- merase 8, has been isolated and designated DNA polymerase III [7].

The cloning and sequencing of the yeast DNA polymerase I gene [8,9], together with the produc-

0167-4781/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

tion of temperature-sensitive mutants [10,11] and a careful examination of the biochemical proper- ties of the enzyme [12], allow the conclusion that DNA polymerase I is the equivalent of DNA polymerase a in yeast cells. Moreover, a DNA primase activity is found to be associated with yeast DNA polymerase I. This bifunctional com- plex consists of four polypeptides, of 180, 74, 58 and 48 kDa [12,13]. The p180 polypeptide is the native core DNA polymerase, while DNA primase is associated with the p58 and p48 protein species [12]. No enzymatic activity has been identified, until now, for the p74 polypeptide. The poly- peptide structure and biochemical properties of polymerase-primase complexes isolated from several eukaryotic organisms is remarkably simi- lar, suggesting that this particular arrangement has been highly conserved during evolution. All the genes encoding the four polypeptides of the yeast complex have now been cloned and se- quenced in our and other laboratories (Refs. 8, 9, 14-16; Foiani et al., unpublished data; Hinkle, D., personal communication) and the analysis of their gene products and the characterization of temperature-sensitive mutants should allow a be- tter definition of their in vivo functions. In this manuscript, we summarize our present knowledge of the structure and function of the yeast DNA polymerase-primase complex. Putative functional domains which are in common between yeast and other DNA polymerases can be identified by se- quence comparison and by the analysis of yeast temperature-sensitive mutants. A highly specific autocatalytic affinity-labeling procedure has also been used to map the active center of yeast DNA primase.

Materials and Methods

Enzymes The yeast DNA polymerase-primase complex

was purified by immunoaffinity chromatography using mouse monoclonal antibodies to yeast DNA polymerase covalently linked to protein-A-Seph- arose [13]. Production of specific antisera against isolated protein subunits and immunodetection of the reactive polypeptides on nitrocellulose filters have been previously described [14].

269

Autocatalytic affinity labeling of DNA primase The p-hydroxybenzaldehyde ester of ATP used

as the affinity labeling reagent was synthesized according to a published procedure [17] and was kindly provided by A.J. Lindner and G.R. Hart- mann (Institut fiir Biochemie, Universitat Miin- chen, F.R.G.). Reactions to affinity-label DNA primase were performed essentially as previously described [18]. A detailed analysis of the mapping of the yeast DNA primase active center is to be described elsewhere (Foiani et al., unpublished data).

Results

Structure and function of the yeast DNA poly- merase-primase complex

The yeast DNA polymerase-primase can be purified in a single step starting from a yeast crude extract by immunoaffinity chromatography, and the same monoclonal column can be used to dissociate DNA primase from DNA polymerase [13]. Polyacrylamide gel electrophoresis of the bi- functional complex in the presence of sodium dodecyl sulfate (SDS-PAGE) reveals four major polypeptides, termed p180, p74, p58 and p48 according to their molecular weight. The produc- tion of specific antisera against these isolated pro- tein species has been essential in establishing the function of the different polypeptides of the com- plex and in cloning their respective genes (Table I). Earlier biochemical and immunochemical stud- ies allowed to correlate the catalytic core DNA polymerase to the p180 polypeptide. In fact, this protein species showed DNA polymerase activity when assayed in situ on SDS-PAGE gel after renaturation of the separated polypeptides, and the enzymatic activity is inhibited by antisera specifically recognizing p180 [12,13]. A body of circumstantial evidence [12] indicated that DNA primase was associated to the p58 and p48 poly- peptides of the yeast complex, but no direct evi- dence was available until now.

Recently, we found that both anti p48 and anti p58 antisera inhibit DNA primase activity, and the degree of inhibition is more evident when primase is dissociated from DNA polymerase (Ref. 14 and unpublished data). This observation sup- ports the finding that the p48 and p58 poly-

270

TABLE I

THE YEAST D N A POLYMERASE-PRIMASE COMPLEX

Subunit Cloned Function Homology (mass, gene kDa) "

180 P O L l D N A polymerase DNA poly- 74 /3 subunit merase ct

gene b unknown 58 P R I 2 c D NA primase 48 P R I 1 D N A primase small subunit

mouse DNA primase '~

a Molecular mass has been determined by SDS-PAGE. b Hinkle, D., personal communication.

Foiani et al., unpublished data. d Tseng, B.Y., personal communication.

peptides are tightly associated and suggests that both subunits might be involved in the DNA- priming reaction.

Recently, a novel methodology has been devel- oped for the highly selective labeling of the active center of prokaryotic and eukaryotic RNA poly- merases by autocatalysis [17-19]. Because DNA primase is a specialized RNA polymerase devoted

to the initiation of DNA synthesis, we applied the same approach to this enzyme, by using the p-hy- droxybenzaldehyde ester of ATP as shown in Fig. 1. The nucleotide derivative can react with nucleophilic amino groups of the protein, and the Schiff base formed can be stabilized by mild re- duction with sodium borohydride. The nucleotide esters that are bound to the primase at or near the active site can participate in phosphodiester bond formation with an incoming radioactive nucleo- tide. Consequently, the enzyme becomes labeled and the active protein subunit(s) can be identified by SDS-PAGE electrophoresis, followed by auto- radiography. As shown in Fig. 1 both the p48 and p58 subunits become labeled with this affinity labeling procedure. The reaction is absolutely de- pendent on the addition of the nucleotide deriva- tive and the template DNA, and it requires reduc- tion with sodium borohydride. Labeling of p48 and p58 is unaffected by DNAase and RNAase treatments performed before gel electrophoresis, while it is completely eliminated by proteinase K treatment (data not shown). These results clearly indicate that both DNA primase subunits par- ticipate in the formation of the active center,

®

® 1 2 3 4

E -I-X ~ EpX N a B H 4

EpX +Io( Z2p] NTP --- EpX~N

I S D S - PAGE

AUTORADIOGRAPHY

E = E n z y m e ( D N A p r i m a s e ) H O O O

X = Nuc leo t ide derivat ive X = c--(( I)-O-P--O--P--O--P--Ado / / ~ i i J

N= L a b e l e d N T P o o- o- o-

Fig. 1. Affinity labeling of yeast DNA primase by autocatalysis. (A) Scheme of the affinity labeling of yeast D N A pnmase with an adenine nucleotide derivative. The chemical structure of the p-hydroxybenzaldehyde ester of ATP is also shown. (B) After the affinity labeling reaction, the polypeptides of the yeast DNA polymerase-primase complex were separated by SDS-PAGE followed by autoradiography. Lane 1, DNA polymerase-primase complex stained with Coomassie blue; lane 2, autoradiogram of the affinity-labeled complex modified with the adenine nucleotide derivative. Samples treated as that analyzed by autoradio- graphy in lane 2 were transferred to nitrocellulose filters and immunoreactive bands were detected with specific anti-p48 (lane 3)

and anti-p58 (lane 4) antibodies.

although the ATP-binding site was localized ex- clusively on the p48 subunit by ultraviolet photo- crosslinking (Foiani et al., unpublished data).

Functional domains in the DNA polymerase I poly- peptide

Highly selective biochemical methods, such as the affinity labeling procedure described above, do not represent the only experimental approach to correlating enzymatic activities with certain polypeptides or to identifying functional domains within a single protein species. Protein sequence comparison among enzymes with analogous func- tions and the characterization and sequencing of available mutants can also be very informative.

The POLl and PRI1 genes of the yeast DNA polymerase-primase complex have been isolated and sequenced in our laboratory and are both essential for cell viability [12,15,16].

Several groups have noted a limited amino-acid sequence homology among DNA polymerases from several animal viruses and phage q~29 [20]. We extended this analysis and, particularly, we compared the amino-acid sequence of the yeast POLl gene product with that derived from cDNA clones encoding the human DNA polymerase ot catalytic polypeptide [21]. As shown in Fig. 2, we found that there is homology in regions I, II and III among all the DNA polymerases listed. These three conserved regions are all included in the carboxy-terminal half of the protein and are found in the same linear arrangement. The function(s) of these conserved regions is still unknown, but the finding that the sites of mutations altering the drug sensitivity of herpes virus DNA polymerase to acyclovir and phosphonoacetic acid fall in re- gions II and III suggests that these regions might define functional domains related to the catalytic site of DNA polymerase [20].

Certainly, the highest degree of homology is found between the yeast and human DNA poly- merase a, as shown by a homology matrix (Fig. 3). The two sequences show 32.7% identity, which is mostly confined to the carboxy-terminal half of the proteins, which includes regions I, II and III. However, a new region of high homology can be identified in the amino-terminal half of the yeast and human DNA polymerases. This region has been named region P, because we believe it de-

Y e a s t 9 8 3 , L A E S N N L L V V Y G D T D S V N I D T G C D N Y A D A

H u m a n 9 8 9 . . . . . N N L - V - Y G D T D S - N 1 - T - - - N . . . .

H S V 8 7 3 . . . . . . . . . . . Y G D T D S . . . . . . . . . . A - -

C N V 8 9 6 . . . . . . . . . V - Y G D T D S V . . . . . . . . . . . .

V Z V 8 3 9 . . . . . . . . . V - Y G D T D S V - I . . . . . . . . . .

E B V 7 4 2 - L . . . . . L - V - Y G D T D S V - I . . . . . . . . . .

V m c c i n i s 7 1 3 . . . . . . . . . . . Y G D T D S V . . . . . . . . . . . .

A d 2 8 3 8 - - - E - - - L - - - Y G D T D S . . . . . . . . . . . . .

p G K L 1 8 6 1 . . . . . . . . . . . Y - D T D S . . . . . . . . . . A - -

S - 1 7 0 4 . . . . . . . . . . . ' - D T D S V . . . . . . . . . . . .

~ 2 9 4 a O - - A . . . . . . . . ' - D T D S . . . . . . . . . . A - -

271

Y e a s t H u m a n H S V

C M V

V Z V

, B Y

, a c c l a i m

A d 2

p G K L 1

5 - 1

~ 2 9

8 4 3 ' Q G G L V F E P E K G L H K N Y V L V N D F H S L Y P S I I Q E F N I C F T T

8 3 9 ' - G G L V - - P - - G . . . . . . L - - D F N S L Y P S I J Q E F N I C F T T

6 9 6 Y Q G - - V - - P - - G - H - N - V - V - D F - S L Y P S I I Q - - , . . . . .

6 9 6 Y Q G - - V F E P E - G . . . . . V - V - D F - S L Y P S I T - - - N - C - - T

6 6 2 Y - G - - - F - P - - , . . . . . V - V - D F - S L Y P , 1 ] Q - - N - C F T T

5 6 3 Y Q G - - V - E P - - G . . . . . V L V - D F - S L Y P S I I Q - - H - C - - T

5 0 3 Y - G G - V F - P . . . . . . N - V L - - D - N S L Y P . . . . . . N . . . . T

5 2 2 - - G G . . . . . . . G . . . . . . . V - D . . . . Y - S . . . . . . . . . . .

6 2 8 . . . . . . . . . . . . . . . . . V L - - D - - S L Y P . . . . . . . . . . . .

4 6 4 . . . . . . . . . . . . . . . . . . . . . D - N S L V P S . . . . . . . . . . T

2 2 5 . . . . . . . . . . K . . . . . . . . V - D - N S L Y P . . . . . . . . . . . .

8[~!9~_~

Y e a s t 9 4 0 Q Q A L K L T A N S N Y G C L G Y V N S R F Y A K P L A N L V T N K G I t E I L

H u m a n 9 4 6 Q - ' L , L T A N S N Y G C L G - - - S R F Y A K P L A - L V T - K G R E 1 L

H S V 8 0 7 Q - A - K - - - N , - , G - - , . . . . . . . . . . . A - - V T - - G R E - L

C N V 8 0 7 Q - A L K - T - N - - Y G - - G - V N . . . . * - P - A - - - T - - G R - - L

V Z V 7 7 1 , - ' - , - - - N S - Y G - - G . . . . . . . . . . . A - - V T - - G R - - L

E B V 6 7 7 , - ' - ' - ' - ' - - ' , - - , - - ' - - , . . . . . A - - V T - - G R - - L

V m c c l n l m 6 3 3 , - - - , - - , , , - , , - - , - - , , - - , . . . . A - - - T - - G R - - -

A d 2 6 8 1 - - A - ' L - - N - - Y , . . . . . . . . . . . . . . . . . . . . . . . . T -

p G K L 1 7 8 0 . . . . ' - - - ' S - - O . . . . . . . . , . . . . . . . . . . . . . . E - -

S - I 6 1 3 . . . . K - T - N S - Y G - - G . . . . . . . . . . . . . . . . . . . . . . .

11129 3 ? 5 . . . . , L - - N , - Y , . . . . . . . . . . . . P . . . . . . . . , . . . .

_~_z919_N_2

Y e a s t 4 6 3 P S D L S S D T F Y H V F G G N S N 1 F E S F V I Q N R I N G P C , L D I K

Human 4 6 5 P - D L - - - T F - H V F G - N . . . . E - F . . . . . I - G P C W L - - K

Fig. 2. Regions of homology in yeast DNA polymerase I and other DNA polymerases. The figure shows the major homolo- gous regions found in the DNA polymerases listed, and the amino acids in common with yeast DNA polymerase I are indicated. A dash indicates no absolute conservation. The numbers indicate the position of the first residue in each sequence. Human, DNA polymerase a from KB cells; HSV, herpes simplex virus; CMV, human cytomegalovirus; VZV, varicella-zoster virus; EBV, Epstein-Barr virus; Vaccinia, vac- cinia virus; Ad2, adenovirus type 2; pGKL1, killer plasmid from K. lactis; S-l, mitochondrial particles from maize; 029, bacteriophage 029. Complete references can be found

in Ref. 16.

fines a functional domain important for protein- protein interactions. In fact, we have recently con- structed yeast poll temperature-sensitive mutants by in vitro mutagenesis of the cloned gene and in vivo replacement of the wild-type allele with the

272

~6 C~l CO

0 0 .

z r~

5 -r

0 250 500 750 1000 1250 I I I I I

, / /

/ "

/

-1250

-1000

-750

- 500

-250

Yeast POLl (1468 a.a.)

Fig. 3. Homology matrix between the amino-acid sequences of

yeast DNA polymerase I and human D N A polymerase a. The

two sequences have been compared using a dot matrix

computer program with the window set at 30 and the strin-

gency at 40.

mutated copy [11]. Preliminary physiological and biochemical characterization of one of these mutants (po l l - l ) indicates that it is a slow-stop DNA synthesis mutant which is defective in the DNA polymerase-primase complex stability [11]. The temperature-sensitive mutation has been se- quenced and it is a single G- C --* A- T transition at position + 1477, which results in the replace- ment of the glycine at codon 493 with arginine [16]. This mutation is included in region P and that glycine is conserved also in human DNA polymerase a. We propose that region P is found in yeast and human DNA polymerase a, because these two proteins are found in a tight complex with DNA primase, and region P might be related to the stability of the polymerase-primase complex found in several eukaryotic cells.

We have recently verified that the CDC17 gene isolated in Hartwell's laboratory [22] is allelic to POLl (unpublished results). Yeast cells carrying the cdc17-1 temperature-sensitive allele quickly stop both growth and DNA synthesis when shifted to the nonpermissive temperature, as expected for a mutation affecting the catalytic activity of the enzyme. By recombinational analysis we mapped the cdc17-1 mutation in the conserved region III

described above (data not shown). These data further support the hypothesis that the conserved sequences in the carboxy-terminal part of all the DNA polymerases shown in Fig. 2 include func- tional domains important for the catalytic activity of eukaryotic DNA polymerases.

Discussion

A DNA polymerase-primase complex has been isolated during the last few years from several eukaryotic organisms and its polypeptide structure appears to be conserved from yeast to mammalian cells [2]. The detailed understanding of the in vivo function and molecular mechanism of this essen- tial component of the DNA replication machinery requires a combination of biochemical and genetic approaches. In this regard, the yeast Sac- charomyces cerevisiae is a particularly attractive as a model system. The yeast DNA polymerase- primase complex is composed of a core DNA polymerase polypeptide (p180), two DNA primase subunits (p48 and p58), and a protein species of unknown function (p74). All the genes coding for these four polypeptides have been cloned and sequenced (see Table I). Cloning of these genes has been particularly informative. In fact, by gene disruption experiments, it has been possible to prove that at least the POLl and PRI1 gene products are essential for cell viability [12]. More- over, we produced and characterized poll condi- tional lethal mutants [11]. The characterization and sequencing of such a. kind of mutant, in conjunction with protein sequence comparison, can be extremely useful in assessing the role of the DNA polymerase-primase complex in the differ- ent aspects of DNA transactions, and in defining the functional domains involved in DNA binding, phosphodiester bond formation and prote in-pro- tein interactions. Amino-acid sequence compari- son between the yeast POLl gene product and several other DNA polymerases revealed three conserved regions of homology (regions I, II and III) that likely include the catalytic site of the enzyme. This conclusion is partially supported by our characterization of a poll temperature-sensi- tive allele (cdc 17-1) whose mutation has been mapped in region III. On the other hand, the poll-1 temperature-sensitive mutation, which ira-

273

pairs the stability of the polymerase-primase com- plex, has been mapped in region P. This region, which is conserved also in the amino-terminal por t ion of human D N A polymerase a, might be impor tant for p ro te in -pro te in interactions be- tween D N A polymerase and D N A primase.

The finding of conserved amino-acid regions in several D N A polymerases might be impor tant to identify genes of unknown functions. Recently, it has been found that the R E V 3 and CDC2 genes code for proteins whose sequences reveal the three conserved regions f o u n d in several D N A polymerases [23,24], raising the possibility that they might represent the structural genes encoding for D N A polymerases II and I I I in S. cerevisiae.

A highly specific affinity-labeling procedure al- lowed to demonst ra te that both primase subunits part icipate in the format ion of the active center of the enzyme. Because the pr imary structure of both polypeptides is known (Ref. 15; Foiani et al., unpublished data), it should be possible, by gener- ating a set of specific proteolytic fragments, to localize the functional site involved in phospho- diester bond format ion at the amino-acid level. These biochemical analysis, together with the pro- duct ion of temperature-sensitive mutants, might allow definition of the functional domains in- volved in the D N A priming reaction.

Acknowledgments

We thank L. Hartwell for gift of strains, F. Grosse for help in computer analysis between yeast and human D N A polymerases, and A.J. Lindner and G.R. H a r t m a n n for their collabora- tion in applying the autocatalytic affinity labeling procedure to yeast D N A primase.

References

1 Bollum, F.J. (1960) J. Biol. Chem. 235, 2399-2403. 2 Kaguni, L.S. and Lehman, I.R. (1988) Biochim. Biophys.

Acta 950, 87-101.

3 So, A.G. and Downey, K.M. (1988) Biochemistry 27, 4591-4595.

4 Kornberg, A. (1982) Supplement to DNA Replication, W.H. Freeman, San Francisco.

5 Wintersberger, U. and Wintersberger, E. (1970) Eur. J. Biochem. 13, 20-27.

6 Chang, L.M.S. (1977) J. Biol. Chem. 252, 1873-1880. 7 Bauer, G.A., Heller, H.M. and Burgers, P.M.J. (1988) J.

Biol. Chem. 263, 917-924. 8 Johnson, L.M., Snyder, M., Chang, L.M.S., Davis, R.W.

and Campbell, J.L. (1985) Cell 43, 369-377. 9 Lucchini, G., Brandazza, A., Badaracco, G., Bianchi, M.

and Plevani, P. (1985) Curr. Genet. 10, 245-252. 10 Budd, M. and Campbell, J.L. (1987) Proc. Natl. Acad. Sci.

USA 84, 2838-2842. 11 Lucchini, G., Mazza, C., Scacheri, E. and Plevani, P. (1988)

Mol. Gen. Genet. 212, 459-465. 12 Plevani, P., Foiani, M., Francesconi, S., Mazza, C., Pizza-

galli, A., Valsasnini, P. and Lucchini, G. (1988) Cancer Cell, Vol. 6, pp. 341-346, Cold Spring Harbor Laboratory, New York.

13 Plevani, P., Foiani, M., Valsasnini, P., Badaracco, G., Cheriathundam, E. and Chang, L.M.S. (1985) J. Biol. Chem. 260, 7102-7107.

14 Lucchini, G., Francesconi, S., Foiani, M., Badaracco, G. and Plevani, P. (1987) EMBO J. 6, 737-742.

15 Plevani, P., Francesconi, S. and Lucchini, G. (1987) Nucleic Acids Res. 15, 7975-7989.

16 Pizzagaili, A., Valsasnini, P., Plevani, P. and Lucchini, G. (1988) Proc. Natl. Acad. Sci. USA 85, 3772-3776.

17 Grachev, M.A., Koiocheva, T.I., Lukhtanov, E.A. and Mustaev, A.A. (1987) Eur. J. Biochem. 163, 113-121.

18 Riva, M., Schaffner, A.R., Sentenac, A., Hartmarm, G.R., Mustaev, A.A., Zaychikov, E.F. and Grachev, M.A. (1987) J. Biol. Chem. 262, 14377-14380.

19 Schaffner, A.R., Jorgensen, E.D., Mc Allister, W.T. and Aartmann, G.R. (1987) Nucleic Acids Res. 15, 8773-8781.

20 Larder, B.A., Kemp, S.D~ and Darley, G. (1987) EMBO J. 6, 169-175.

21 Wong, S.W., Wahl, A.F., Yuan, P.M., Arai, N., Pearson, B.E., Arai, K., Korn, D., Hemkapiller, M.W. and Wang, T.S.F. (1988) EMBO J. 7, 37-47.

22 Carson, M.J. and Hartwell, L. (1985) Cell 42, 249-257. 23 Morrison, A., Lemontt, J.F., Beck, A.K., Bernstine, E.G.,

Christens, R.B., Banerjee, S.K. and Lawrence, C.W. (1988) Yeast 4, S130.

24 Boulet, A., Simon, M. and Faye, G. (1988) Yeast, 4, Sl19.