Moi Gen Genet (1992) 235:450~452 © Springer-Verlag 1992
Short communications
The DAC2/FUS3 protein kinase is not essential for transcriptional activation of the mating pheromone response pathway in Saccharomyces cerevisiae Hiro-aki Fujimura Laboratory for Molecular Biology,Pharma Research Laboratories, Hoechst Japan Limited, 1-3-2 Minamidai, Kawagoe 350, Japan Received April 18, 1992 / Accepted May 4, 1992
Summary. The DAC2/FUS3 gene of Saccharomyces cerevisiae, which encodes a CDC28/cdc2-related protein kinase, is essential both for the arrest of cell division induced by mating pheromones and for cell fusion during conjugation. To elucidate the role of the DAC2 gene product in the pheromone response pathway, I determined the nucleotide sequence of the DAC2 gene and characterized two types of deletion mutants of the DAC2 gene. Here, I show that the DAC2 gene is identical to the FUS3 gene and that dac2/fus3 deletion mutants respond to mating pheromones by activating transcription. Therefore, the DAC2/FUS3 gene is not essential for transcriptional activation in the pheromone response pathway. The DAC2/FUS3 protein kinase has a positive role in cell fusion during sexual conjugation. Key words: Pheromone response pathway - Signal transduction - Transcription - Protein kinase - Yeast DAC2/ FUS3 gene
The yeast mating pheromones, termed a-factor and afactor, control the growth and differentiation of haploid cells of opposite mating types during conjugation in Saccharomyces cerevisiae (for reviews see Cross et al. 1988; Herskowitz 1989). Pheromones bind to receptors on the target cells of opposite mating type to induce arrest of cell division in the G1 phase, production of cell surface agglutinin, morphological changes, and transcriptional activation of specific genes required for conjugation. Molecular and genetic analysis has revealed that many gene products are involved in the pheromone response pathway. Recently, Elion et al. (1990) identified the FUS3 gene, which was necessary both for cell division arrest at G1 phase and for transcriptional activation in response to a-factor in Saccharornyces cerevisiae. The fus3 I mutation was originally isolated in a screen for mutants defective in cell fusion during conjugation; it also causes resistance to a-factor. Null alleles of FUS3 (creating by
insertion of exogenous DNA or by deletion) caused cells to become completely unresponsive to a-factor. This result predicts that FUS3 has at least two functions: to activate transcription, possibly by acting on STE12, and to inactivate CLN3. However, the dual phenotype also makes the role of the FUS3 gene in cell division arrest more difficult to interpret. Thus, deletion of the FAR1 gene, which is required to inhibit CLN2 leads to a specific defect in cell division arrest (Chang and Herskowitz 1990; Nasmyth 1990). On the other hand, I have identified the DAC2 gene, which is necessary for cell division arrest by a-factor (Fujimura 1990a; Fujimura 1990b). Comparison of the restriction enzyme maps of the DAC2 and FUS3 genes indicates that DAC2 may be the same gene as FUS3. In fact, fus3 maps to the right arm of chromosome II, just near the dac2 locus. However, there is one clear difference between DAC2 and FUS3: the null allele of DAC2 (created by an insertional mutation) does not affect multiple responses to a-factor; the dac2 null mutant is defective only in cell division arrest. In this study I determined the nucleotide sequence of the DAC2 gene to establish whether or not the DAC2 gene is identical to the FUS3 gene. In addition, I constructed two types of deletion mutations in the DAC2 gene and examined the response of these mutants to a-factor in order to reach a definitive conclusion on the role of the DAC2/FUS3 gene product in the pheromone response pathway. Plasmid pDAC2-E3, containing the entire sequence of the DAC2 gene (Fujimura 1990b) was digested with various restriction enzymes, and the resulting fragments were subcloned into pUC 13. DNA sequence analysis was carried out using the dideoxynucleotide chain termination method (Sanger et al. 1977). As shown in Fig. 1, the nucleotide and deduced amino acid sequences of the DAC2 gene were completely identical to those of the FUS3 gene (Elion et al. 1990), except that amino acid 334 was tyrosine instead of histidine, and that the codons for amino acids 47, 310 and 316 were TTC, CAA and AAC instead of TTT, CAG and AAT, respectively. DNA
451 1
AACTGGAGAGGGCAGTTTACTTT CACATACTCTTTTATCTTTCTGAAGTAAAATGTGATGTACCTACAAGC~TGCATAAGCTCTTGAGT
91
TTTAGATCTGTTTCACCCTGGAACTCAAAATTCTTTTACTCGAAATTTTTACTTTTTTTTTTTTTGTTTCTGCATTCTCTCAGATTTTAG
181
ATGATGCGGTTTTTTACAGGGCATTGAAACAATTGCAG~CAACATACTAATATAT CATAACTTTTTACTCTTGCCTCTCAGAAACT
271
ATATATACGTTGTAATCATTTTCTTTCTTCTAATAGCTAGTTCGTTTGAACTACAAGGAAATAAGGCAGAGAAAAAGAAAGGAAAATAAT
361 1
ATGCCAAAGAGAATTGTATAC, AATATATCCAGTGACTTCCAGTTGAAGTC.GTTACTGGGAGAGGGTGCATACGGTGTGGTATGTTCTGCA M P K R I V Y N I S S D F Q L K S L L G E G A Y G V V C S A
451 31
ACGCATAAGCCCACGGGAGAAATCGTGGCAATAAAAAAGAT CGAACCATTCGATAAGCCTTTGTTCGCATTACGTACGCTGCGTGAAATA T H K P T G E I V A I K K I E P F D K P L F A L R T L R E I
541 61
AAGATCCTGAAGCACTTCAAGCACGf~.A.A.A.'FATCATAACAATCTTCAACATTCAACGCCCTGACTCGTTCGAAAACTTCAATGAGGTGTAC K I L K H F K H E N I I T I F N I Q R P D S F E N F N E V Y
631 91
ATAATTCAAGAGCTAATGCAGACAGATTTACACC,;GTGTAAT CTCCACCCAGATGCTGAGTGACGATCATATACAATATTTTATATACCAA I I Q E L M El T D L H R V I S T Q M L $ D D H I Q Y F I Y El
721 121
ACCTTGAGAGCAGTGAAAGTGCTGCATGGTTCGAACGT CATCCATCGTGATTTAAAGCCCTCCAACCTTCTCATAAACTCCAACTGTGAC T L R A V K V L H G S N V I H R D L K P S N L L I N S N C D
811 151
TTGAAAGTATGTGATTTCGGTTTAGC/~'AGAATCATTGACGAGT~AGCCGCGGACAATTCAGAGC~CACAGGTCAGCAAAGCGGCATGA~ L K V C D F G L A R I I D E S A A D N S E P T G El 0 S G M T
901 181
GAGTATGTGGCCA•ACGTTGGTACAGGGCGCCAGAGGTGATGTTAACCT•TGCCAAATA•TCAAGGG•CATGGACGTGTGGT CCTGCGGA E Y V A T R W Y R A P E V M L T S A K Y S R A M D V W S C (3
991 211
TGTATTCTCGCTGAACTTTTCTTAAGACGGCCAATCTTCCCTGGCAGAGATTATCGCCATCAACTACTACTGATATTCGGTATCATCGGT C I L A E L F L R R P I F P O R O Y R H El L L L I F G I I O
1081 ACA•CTCA•T•AGATAATGATTT•CGGTGTATAGAGTCA•C•AGGGCTAGAGAGTACATAAAGTcGCTTC•CATGTA•••TG•CGCGC•A 241 T P H S D N D L R C I E S P R A R E Y I K S L P M Y P A A 1171 271
P
CT•GAGAAGATGTTCCCTCGAGTCAACCCGAAAGGCATA•ATCTTTTACAGCGTATGCTTGTTTTTGACCCTG•••GAAGAGGATTACTGCT L E K M F P R V N P K G I D L L El R M L V F D P A K R I T A
1261 AAGGAGG•ACTGGAG•ATCCGTATTTGCAAA•ATACCACGATCCAAA•GAcGAAC•TGAAGG•GAA•CCAT•C•AC•CAGCTTCTT•GAG 301 K E A L E H P Y L El T Y H D P N D E P E G E P I P P S F F
E
1351 TTTGATCACTACAAGGAGGCACTAACGACGAAAGACCT CAAGAAACTCATTTGGAACGAAATATTTAGTTAGCCATCATTATCATTAAAA 331 F D H Y K E A L T T K D L K K L I W N E I F S 1441
TATCAACCCGAAGAACAATGTATACATATACATATACGTACACATATACATATGTACATATGACATACGTATTA
H ,
X Nd XXmNc I I II I
I H
X
Xm I
B ,
= X Nc
L NdXm
pDAC2-E3
pDAC2 - a 4LEU2
( Xm
I B
p D A C 2 - a 5LEU2
Fig. 2. Structure o f d i s r u p t i o n plasmids, p D A C 2 - A 4 L E U 2
and
pDAC2-A5LEU2 were constructed as described in the text. The coding region for the DAC2 gene is indicated by the arrow. The closed bar represents yeast genomic sequence. Restriction sites: B, BarnHI; H, HindIII; Nc, NcoI; Nd, NdeI; X, XhoI; Xm, XmnI
sequencing analysis revealed the presence of the a1-{,2 consensus element (Miller et al. 1985) in the 5' flanking region of the DA C2 gene. This element is found in the upstream region of genes, that are specifically expressed in haploid cells such as FUS1 and SST2, (Trueheart et al. 1987, McCaffrey et al. 1987, Dietzel and Kurjan 1987). The pheromone response element, TGAAACA, was also found in the 5' flanking region of the DAC2 gene, just as in the FUS3 gene (Van Arsdell et al. 1987, Elion et al. 1990). A dac2 deletion mutation (dac2-A4.':LEU2) was created by the removal of an 0.6 kb XhoI fragment from the DA C2/FUS3 gene on plasmid pDAC2-E3 (Fujimura 1990b), which was replaced by the LEU2 gene on a 2.2 kb SalI-XhoI fragment (pDAC2-A4LEU2). A second dac2 deletion mutation (dac2-A5 : :LEU2) was created by the removal of a 0.7 kb XmnI fragment followed by the substitution of the 2.2 kb LEU2 gene (pDAC2-A5LEU2; Fig. 2). Each dac2/fus3 disruption plasmid was digested
Fig. 1. Nucleotide and deduced amino acid sequences of the DAC2 gene. The proposed coding region is shown with the corresponding amino acids below. Thick underlining indicates the sequence similar to the al-ct2 consensus element (Miller et al. 1985). Sequences related to the pheromone
response dement consensus, TGAAACA, are indicated by thin underlining (Van Arsdell et al. 1987)
with NcoI and HindIII or NdeI and BamHI to liberate a fragment suitable for integration into yeast by one-step gene disruption (Rothstein 1983). A diploid strain XF 106 (MATa/MATa ura3/ura3 leu2/leu2 his4~+ +/trpl gal2/ + ; Fujimura 1990b) was transformed with digested pDAC2-A4LEU2 or pDAC2-A5LEU2 using the lithium method (Ito et al. 1983). Leu + transformants were sporulated, and their meiotic segregants were analyzed for growth and ability to mate by standard methods (Sherman et al. 1983). Tetrad analysis showed 2:2 cosegregation of leucine prototrophy and sterility. To determine whether the DAC2/FUS3 gene is essential for transcriptional activation charasteristic of the pheromone response, a 2 btm-based plasmid, pSB234, containing a FUSI-lacZ fusion gene (Trueheart et al. 1987), was introduced into wild-type cells and dac2/fus3 deletion mutants, and [3-galactosidase activity was examined after a-factor treatment. As shown in Table 1, the level of FUS1 transcript, as estimated from the [3-galac-
Table 1. Induction of expression of FUSI-lacZ fusion gene by {,-factor Strain a
XF116-5A XFll4-2B XFll6-3A
Relevant genotype
Units of [3-galactosidase activity b -- c~- factor
DAC2 0.91 dac2-k4:: LEU2 7.4 dac2-kS:: LF_.U2 3.0
+ a - factor ° 173 146 135
a All strains are MATa and carry the plasmid pSB234 (Trueheart et al. 1987). pSB234 is a 2 gm-URA3 based plasmid which encodes a FUSI-lacZ fusion product (the fusion contains the first 254 amino acids of FUS1 fused to [3-galactosidase) b Units of activity were determined according to Ausubel et al. (1989) ° c~-Factor (1.7 gg/ml) was added to each cell culture
452
References
Fig. 3. Zygote formation. Cells were allowed to mate in YEPD (Sherman et al. 1983) for 6 h at 30° C. A sample was then viewed with a phase-contrast microscope. X F l l 6 - 3 A (MATa dac2A5: :LEU2) x XF64-54D (MATe0
tosidase activity, was similar to that observed when wildtype cells were treated with a-factor. This result indicates that FUS1 transcription is rapidly increased by s-factor treatment in the dac2/fus3 deletion mutants, just as in wild-type cells, and therefore the DAC2/FUS3 gene is not essential for transcriptional activation of the pheromoneinducible FUS1 gene. The dac2 deletion mutants also showed morphological changes in response to a-factor. Thus, I could not confirm the finding of Elion et al. (1990) indicating thatfus3 insertion and deletion mutants are unresponsive to a-factor and do not undergo morphological changes, although the original fus3-1 and fus3-2 mutants are responsive. The behavior of dac2 deletion mutants cultured in the presence of wild-type cells of opposite mating type was also investigated. The dac2 cells agglutinated and formed prezygotes with the wild-type cells, but could not complete cell fusion (Fig. 3), suggesting that the DAC2/FUS3 gene product may indeed play a positive role in cell fusion during conjugation as suggested previously (Elion et al. 1990; Fujimura 1990a; Fujimura 1990b).
Acknowledgments. I thank J. Trueheart for providing me with plasmid pSB234.
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1989) Current protocols in molecular biology. Wiley Interscience and Green Publishing Associates, New York Chang F, Herskowitz I (1990) Identification of a gene necessary for cell cycle arrest by a negative growth factor of yeast: FARI is an inhibitor of a G1 cyclin, CLN2. Cell 63 : 999-1011 Cross F, Hartwell LH, Jackson C, Konopka JB (1988) Conjugation in Saeeharomyees eerevisiae. Annu Rev Cell Biol 4:429-457 Dietzel C, Kurjan J (198"]) Pheromonal regulation and sequence of the Saeeharomyees eerevisiae SST2 gene: a model for desensitization to pheromone. Mol Cell Biol 7:4169-4177 Elion EA, Orisafi PL, Fink GR (1990) FUS3 encodes a cdc2+/ CDC28-related kinase required for the transition from mitosis into conjugation. Cell 60:649-664 Fujimura H (1990a) Identification and characterization of a mutation affecting the division arrest signaling of the pheromone response pathway in Saeeharomyees eerevisiae. Genetics 124:2"]5-282 Fujimura H (1990b) Molecular cloning of the DAC2/FUS3 genc essential for pheromone-induced G1 arrest of the cell cycle in Saeeharomyees eerevisiae. Curr Genet 18:395-400 Herskowitz I (1989) A regulatory hierarchy for cell specialization in yeast. Nature 342:749-757 Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163-168 McCaffrey G, Clay FJ, Kelsay K, Sprague GF Jr (198"]) Identification and regulation of a gene required for cell fusion during mating of the yeast Saeeharomyces eerevisiae. Mol Cell Biol 7: 2680-2690 Miller AM, MacKay VL, Nasmyth KA (1985) Identification and comparison of two sequence elements that confer cell-type specific transcription in yeast. Nature 314:598-603 Nasmyth KA (1990) FAR-reaching discoveries about the regulation of start. Cell 63 : 1117-1120 Rothstein R (1983) One-step gene disruption in yeast. Meth Enzytool 101:202-211 Sanger F, Nicklen S, Coulson AR 0977) DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci USA 74:5463 546"] Sherman F, Fink GR, Hicks JB (1983) Methods in yeast genetics: laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Trueheart J, Boeke JD, Fink OR (1987) Two genes required for cell fusion during yeast conjugation: evidence for a pheromoneinduced surface protein. Mol Cell Biol 7: 2316-2328 Van Arsdell SW, Stetlcr GL, Thorner J (1987) The yeast repeated element sigma contains a hormone-inducible promoter. Mol Cell Biol "]:749-759
Communicated by W. Oajewski
Short communications
The DAC2/FUS3 protein kinase is not essential for transcriptional activation of the mating pheromone response pathway in Saccharomyces cerevisiae Hiro-aki Fujimura Laboratory for Molecular Biology,Pharma Research Laboratories, Hoechst Japan Limited, 1-3-2 Minamidai, Kawagoe 350, Japan Received April 18, 1992 / Accepted May 4, 1992
Summary. The DAC2/FUS3 gene of Saccharomyces cerevisiae, which encodes a CDC28/cdc2-related protein kinase, is essential both for the arrest of cell division induced by mating pheromones and for cell fusion during conjugation. To elucidate the role of the DAC2 gene product in the pheromone response pathway, I determined the nucleotide sequence of the DAC2 gene and characterized two types of deletion mutants of the DAC2 gene. Here, I show that the DAC2 gene is identical to the FUS3 gene and that dac2/fus3 deletion mutants respond to mating pheromones by activating transcription. Therefore, the DAC2/FUS3 gene is not essential for transcriptional activation in the pheromone response pathway. The DAC2/FUS3 protein kinase has a positive role in cell fusion during sexual conjugation. Key words: Pheromone response pathway - Signal transduction - Transcription - Protein kinase - Yeast DAC2/ FUS3 gene
The yeast mating pheromones, termed a-factor and afactor, control the growth and differentiation of haploid cells of opposite mating types during conjugation in Saccharomyces cerevisiae (for reviews see Cross et al. 1988; Herskowitz 1989). Pheromones bind to receptors on the target cells of opposite mating type to induce arrest of cell division in the G1 phase, production of cell surface agglutinin, morphological changes, and transcriptional activation of specific genes required for conjugation. Molecular and genetic analysis has revealed that many gene products are involved in the pheromone response pathway. Recently, Elion et al. (1990) identified the FUS3 gene, which was necessary both for cell division arrest at G1 phase and for transcriptional activation in response to a-factor in Saccharornyces cerevisiae. The fus3 I mutation was originally isolated in a screen for mutants defective in cell fusion during conjugation; it also causes resistance to a-factor. Null alleles of FUS3 (creating by
insertion of exogenous DNA or by deletion) caused cells to become completely unresponsive to a-factor. This result predicts that FUS3 has at least two functions: to activate transcription, possibly by acting on STE12, and to inactivate CLN3. However, the dual phenotype also makes the role of the FUS3 gene in cell division arrest more difficult to interpret. Thus, deletion of the FAR1 gene, which is required to inhibit CLN2 leads to a specific defect in cell division arrest (Chang and Herskowitz 1990; Nasmyth 1990). On the other hand, I have identified the DAC2 gene, which is necessary for cell division arrest by a-factor (Fujimura 1990a; Fujimura 1990b). Comparison of the restriction enzyme maps of the DAC2 and FUS3 genes indicates that DAC2 may be the same gene as FUS3. In fact, fus3 maps to the right arm of chromosome II, just near the dac2 locus. However, there is one clear difference between DAC2 and FUS3: the null allele of DAC2 (created by an insertional mutation) does not affect multiple responses to a-factor; the dac2 null mutant is defective only in cell division arrest. In this study I determined the nucleotide sequence of the DAC2 gene to establish whether or not the DAC2 gene is identical to the FUS3 gene. In addition, I constructed two types of deletion mutations in the DAC2 gene and examined the response of these mutants to a-factor in order to reach a definitive conclusion on the role of the DAC2/FUS3 gene product in the pheromone response pathway. Plasmid pDAC2-E3, containing the entire sequence of the DAC2 gene (Fujimura 1990b) was digested with various restriction enzymes, and the resulting fragments were subcloned into pUC 13. DNA sequence analysis was carried out using the dideoxynucleotide chain termination method (Sanger et al. 1977). As shown in Fig. 1, the nucleotide and deduced amino acid sequences of the DAC2 gene were completely identical to those of the FUS3 gene (Elion et al. 1990), except that amino acid 334 was tyrosine instead of histidine, and that the codons for amino acids 47, 310 and 316 were TTC, CAA and AAC instead of TTT, CAG and AAT, respectively. DNA
451 1
AACTGGAGAGGGCAGTTTACTTT CACATACTCTTTTATCTTTCTGAAGTAAAATGTGATGTACCTACAAGC~TGCATAAGCTCTTGAGT
91
TTTAGATCTGTTTCACCCTGGAACTCAAAATTCTTTTACTCGAAATTTTTACTTTTTTTTTTTTTGTTTCTGCATTCTCTCAGATTTTAG
181
ATGATGCGGTTTTTTACAGGGCATTGAAACAATTGCAG~CAACATACTAATATAT CATAACTTTTTACTCTTGCCTCTCAGAAACT
271
ATATATACGTTGTAATCATTTTCTTTCTTCTAATAGCTAGTTCGTTTGAACTACAAGGAAATAAGGCAGAGAAAAAGAAAGGAAAATAAT
361 1
ATGCCAAAGAGAATTGTATAC, AATATATCCAGTGACTTCCAGTTGAAGTC.GTTACTGGGAGAGGGTGCATACGGTGTGGTATGTTCTGCA M P K R I V Y N I S S D F Q L K S L L G E G A Y G V V C S A
451 31
ACGCATAAGCCCACGGGAGAAATCGTGGCAATAAAAAAGAT CGAACCATTCGATAAGCCTTTGTTCGCATTACGTACGCTGCGTGAAATA T H K P T G E I V A I K K I E P F D K P L F A L R T L R E I
541 61
AAGATCCTGAAGCACTTCAAGCACGf~.A.A.A.'FATCATAACAATCTTCAACATTCAACGCCCTGACTCGTTCGAAAACTTCAATGAGGTGTAC K I L K H F K H E N I I T I F N I Q R P D S F E N F N E V Y
631 91
ATAATTCAAGAGCTAATGCAGACAGATTTACACC,;GTGTAAT CTCCACCCAGATGCTGAGTGACGATCATATACAATATTTTATATACCAA I I Q E L M El T D L H R V I S T Q M L $ D D H I Q Y F I Y El
721 121
ACCTTGAGAGCAGTGAAAGTGCTGCATGGTTCGAACGT CATCCATCGTGATTTAAAGCCCTCCAACCTTCTCATAAACTCCAACTGTGAC T L R A V K V L H G S N V I H R D L K P S N L L I N S N C D
811 151
TTGAAAGTATGTGATTTCGGTTTAGC/~'AGAATCATTGACGAGT~AGCCGCGGACAATTCAGAGC~CACAGGTCAGCAAAGCGGCATGA~ L K V C D F G L A R I I D E S A A D N S E P T G El 0 S G M T
901 181
GAGTATGTGGCCA•ACGTTGGTACAGGGCGCCAGAGGTGATGTTAACCT•TGCCAAATA•TCAAGGG•CATGGACGTGTGGT CCTGCGGA E Y V A T R W Y R A P E V M L T S A K Y S R A M D V W S C (3
991 211
TGTATTCTCGCTGAACTTTTCTTAAGACGGCCAATCTTCCCTGGCAGAGATTATCGCCATCAACTACTACTGATATTCGGTATCATCGGT C I L A E L F L R R P I F P O R O Y R H El L L L I F G I I O
1081 ACA•CTCA•T•AGATAATGATTT•CGGTGTATAGAGTCA•C•AGGGCTAGAGAGTACATAAAGTcGCTTC•CATGTA•••TG•CGCGC•A 241 T P H S D N D L R C I E S P R A R E Y I K S L P M Y P A A 1171 271
P
CT•GAGAAGATGTTCCCTCGAGTCAACCCGAAAGGCATA•ATCTTTTACAGCGTATGCTTGTTTTTGACCCTG•••GAAGAGGATTACTGCT L E K M F P R V N P K G I D L L El R M L V F D P A K R I T A
1261 AAGGAGG•ACTGGAG•ATCCGTATTTGCAAA•ATACCACGATCCAAA•GAcGAAC•TGAAGG•GAA•CCAT•C•AC•CAGCTTCTT•GAG 301 K E A L E H P Y L El T Y H D P N D E P E G E P I P P S F F
E
1351 TTTGATCACTACAAGGAGGCACTAACGACGAAAGACCT CAAGAAACTCATTTGGAACGAAATATTTAGTTAGCCATCATTATCATTAAAA 331 F D H Y K E A L T T K D L K K L I W N E I F S 1441
TATCAACCCGAAGAACAATGTATACATATACATATACGTACACATATACATATGTACATATGACATACGTATTA
H ,
X Nd XXmNc I I II I
I H
X
Xm I
B ,
= X Nc
L NdXm
pDAC2-E3
pDAC2 - a 4LEU2
( Xm
I B
p D A C 2 - a 5LEU2
Fig. 2. Structure o f d i s r u p t i o n plasmids, p D A C 2 - A 4 L E U 2
and
pDAC2-A5LEU2 were constructed as described in the text. The coding region for the DAC2 gene is indicated by the arrow. The closed bar represents yeast genomic sequence. Restriction sites: B, BarnHI; H, HindIII; Nc, NcoI; Nd, NdeI; X, XhoI; Xm, XmnI
sequencing analysis revealed the presence of the a1-{,2 consensus element (Miller et al. 1985) in the 5' flanking region of the DA C2 gene. This element is found in the upstream region of genes, that are specifically expressed in haploid cells such as FUS1 and SST2, (Trueheart et al. 1987, McCaffrey et al. 1987, Dietzel and Kurjan 1987). The pheromone response element, TGAAACA, was also found in the 5' flanking region of the DAC2 gene, just as in the FUS3 gene (Van Arsdell et al. 1987, Elion et al. 1990). A dac2 deletion mutation (dac2-A4.':LEU2) was created by the removal of an 0.6 kb XhoI fragment from the DA C2/FUS3 gene on plasmid pDAC2-E3 (Fujimura 1990b), which was replaced by the LEU2 gene on a 2.2 kb SalI-XhoI fragment (pDAC2-A4LEU2). A second dac2 deletion mutation (dac2-A5 : :LEU2) was created by the removal of a 0.7 kb XmnI fragment followed by the substitution of the 2.2 kb LEU2 gene (pDAC2-A5LEU2; Fig. 2). Each dac2/fus3 disruption plasmid was digested
Fig. 1. Nucleotide and deduced amino acid sequences of the DAC2 gene. The proposed coding region is shown with the corresponding amino acids below. Thick underlining indicates the sequence similar to the al-ct2 consensus element (Miller et al. 1985). Sequences related to the pheromone
response dement consensus, TGAAACA, are indicated by thin underlining (Van Arsdell et al. 1987)
with NcoI and HindIII or NdeI and BamHI to liberate a fragment suitable for integration into yeast by one-step gene disruption (Rothstein 1983). A diploid strain XF 106 (MATa/MATa ura3/ura3 leu2/leu2 his4~+ +/trpl gal2/ + ; Fujimura 1990b) was transformed with digested pDAC2-A4LEU2 or pDAC2-A5LEU2 using the lithium method (Ito et al. 1983). Leu + transformants were sporulated, and their meiotic segregants were analyzed for growth and ability to mate by standard methods (Sherman et al. 1983). Tetrad analysis showed 2:2 cosegregation of leucine prototrophy and sterility. To determine whether the DAC2/FUS3 gene is essential for transcriptional activation charasteristic of the pheromone response, a 2 btm-based plasmid, pSB234, containing a FUSI-lacZ fusion gene (Trueheart et al. 1987), was introduced into wild-type cells and dac2/fus3 deletion mutants, and [3-galactosidase activity was examined after a-factor treatment. As shown in Table 1, the level of FUS1 transcript, as estimated from the [3-galac-
Table 1. Induction of expression of FUSI-lacZ fusion gene by {,-factor Strain a
XF116-5A XFll4-2B XFll6-3A
Relevant genotype
Units of [3-galactosidase activity b -- c~- factor
DAC2 0.91 dac2-k4:: LEU2 7.4 dac2-kS:: LF_.U2 3.0
+ a - factor ° 173 146 135
a All strains are MATa and carry the plasmid pSB234 (Trueheart et al. 1987). pSB234 is a 2 gm-URA3 based plasmid which encodes a FUSI-lacZ fusion product (the fusion contains the first 254 amino acids of FUS1 fused to [3-galactosidase) b Units of activity were determined according to Ausubel et al. (1989) ° c~-Factor (1.7 gg/ml) was added to each cell culture
452
References
Fig. 3. Zygote formation. Cells were allowed to mate in YEPD (Sherman et al. 1983) for 6 h at 30° C. A sample was then viewed with a phase-contrast microscope. X F l l 6 - 3 A (MATa dac2A5: :LEU2) x XF64-54D (MATe0
tosidase activity, was similar to that observed when wildtype cells were treated with a-factor. This result indicates that FUS1 transcription is rapidly increased by s-factor treatment in the dac2/fus3 deletion mutants, just as in wild-type cells, and therefore the DAC2/FUS3 gene is not essential for transcriptional activation of the pheromoneinducible FUS1 gene. The dac2 deletion mutants also showed morphological changes in response to a-factor. Thus, I could not confirm the finding of Elion et al. (1990) indicating thatfus3 insertion and deletion mutants are unresponsive to a-factor and do not undergo morphological changes, although the original fus3-1 and fus3-2 mutants are responsive. The behavior of dac2 deletion mutants cultured in the presence of wild-type cells of opposite mating type was also investigated. The dac2 cells agglutinated and formed prezygotes with the wild-type cells, but could not complete cell fusion (Fig. 3), suggesting that the DAC2/FUS3 gene product may indeed play a positive role in cell fusion during conjugation as suggested previously (Elion et al. 1990; Fujimura 1990a; Fujimura 1990b).
Acknowledgments. I thank J. Trueheart for providing me with plasmid pSB234.
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Communicated by W. Oajewski
