YEASI
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VOL. 7: 651-655 (1991)
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Yeast Sequencing Reports
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The Open Reading Frame YCRlOl located on Chromosome I11 from Saccharomyces cerevisiae is a Putative Protein Kinase JACEK SKALA*,B ~ N E D I C T EPURNELLE, MARC CROUZET', MICHEL AIGLE+A N D ANDREGOFFEAU
Unit6 de Biochimie Phpsiologique, UniversitP Cutholique de Louvain,B- 1348 Louvain-la-Neuve,Belgium 'Lahoratoire de BiolocqieMolkculaire et de SPquenqage, Universitt de Bordeuux 11, 146 rue L t o Saignut, 33076 Bordeaux-cedex, France
Received 30 May 1991; accepted 4 June 1991
KEY WORDSxhromosome 111; sequencing; gene distruption; kinase.
As part of the European project of sequencing the yeast Saccharomyces cerevisiue genome, we have sequenced the DlOH and J 1 ID fragments (Palzkill et a/., 1986) of the 524-24a strain (Strathem etal., 1979). Sequencing was carried out on both strands by the dideoxy chain-termination method (Sanger et a/.. 1977). The DlOH and J l l D fragments are contiguous; their junction has been verified by sequencing 1500 bp of the overlapping clone lambda CH4Ap17 from the strain AB972 (Olson et al., 1986). The 1809 bp long open reading frame YCRlOl has been identified at the junction of DlOH and J11D. YCRIOl is located between the SUF2 (Mortimer et d., 1989) and the KVS161 genes (Urdaci et al., 1990) o n the right arm of chromosome 111. The DNA and deduced amino acid sequences are given in Figure 1. ANALYSIS OF THE 5'- AND 3'-NON-CODING REGIONS Two CAAT boxes have been found in the 5'-noncoding region at positions -418 to -415 and -21 1 to -208, as well as two TATA-like sequences at positions -387 to -383 and -211 to -208 (Figure 1). In the 3'-non-coding region a polyadenylation site consensus
AATAAA (Zaret and Sherman, 1982) at position +2043 to +2047 and a termination site consensus TTTTATA (Henikoff and Cohen, 1984) at position + 1940 to + 1946 were detected (Figure 1). ANALYSIS OF THE OPEN READING FRAME The predicted protein comprises 603 amino acids and has a molecular weight of 66,665 Da. The value of codon bias index calculated according to Bennetzen and Hall (1982) is 0.084 indicating a low level of expression. The corresponding mRNA has been characterized by Yoshikawa and Isono (1990). who estimated that the transcript had a relative abundance of 2 in a scale from 1 (lowest) to 5 (highest abundance). Determination of secondary structure using the method of Gamier et a/. (1978) suggests the existence of two domains in the predicted protein. The aminoterminal domain (residues 1-258) present largely undefined structures while the carboxy-terminal domain (residues 259-603) is highly structured in alpha-helices. The first domain is rich in serine residues (18.2%) compared to the second one (6.3%) and has no significant homology with any protein contained in the data bank.
Editorial correspondence to: Andre Goffeau TEL: 32/10/47 36 14 FAX: 32/10/47 38 72 'Permanent address: Institute of Microbiology, Wroclaw University, Przybyszewskiego 63, 5 1-148 Wroclaw, Poland.
0749-S03X/9 1/060643-08$05.00 01991 by John Wiley & Sons Ltd
652
J. SKALA E T A L
-480 CAGCTTTTAAAGAGGGAAGAGGAAACGGAGAAGAAAATATACTACTTTATAGAGATATTC CAAT
TATA
-420 GGCAATTTTTTTTAGCAAAAGTTTATTAAACCCTAATATAGACCTTTACTTGGAGTTTCG -360 ATTTTAAGAATTTTTTATCATTTTTTTTTTTTGACGATTTCTGTCGGTTTCGTTATAACC -300 TGTTGTGTTGTTGTTGTTGTTGTTATTGCTGGGTTGTTGTTATTCACTTTAACATTATCG CAAT
-240 AATAAAATTTGGACTTTCAAAGTCCTGTTCAATAAGTTGTACTAAGCCTTTAAGCGTTTT H i ndI I I
TATA
-180 TAAGTTACAAATAAAAACTGGAAAGCTTAAGGTAGGTAGCTTTCTAATATTAGGCTCTCAACCT -120 TTTTTTACTCTCCATTTACCGCTAGATATTAACTAGTAGTAAGTATTGTGACTACTATTC -60 CCTGTAATCAAAAAAAAAGTAATCAGATTTTATTTTATTTCGACATTACCCCTCAAATAT
+ I ATGACTGGTATGAATGATAATAATGCCGCTATTCCTCAGCAAACTCCAAGGAAACATGCG M
T
G
M
N
D
N
N
A
A
I
P
Q
Q
T
P
R
K
H
A
20
+61 CTATCTTCTAAAGTTATGCAACTTTTTAGAAGCGGTTCAAGATCATCTAGGCAGGGAAAG L S S K V M Q L F R S G S R S S R Q G K 40 +I21 GCCTCATCGAATATCCAGCCACCTTCTAATATAAACACAAACGTTCCATCGGCGTCTAAA A S S N I Q P P S N I N T N V P S A S K 60
+I81 TCAGCCAAATTTGGTTTACATACCCCAACCACTGCTACTCCTAGGGTAGTTTCTAATCCT S A K F G L H T P T T A T P R V V S N P
80
+241 TCTAATACTGCAGGTGTGAGTAAACCGGGCATGTATATGCCCGAATATTACCAGTCGGCA S N T A G V S K P G M Y M P E Y Y Q S A I O
O
+301 TCACCATCGCACTCTAGTTCATCCGCATCATTAAACAACCATATTGATATTAACACCTCT S P S H S S S S A S L N N H I D I N T S l 2
0
+361 AAGTCATCATCAGCTGCTTCTTTAACTTCGTCAGTATCAGCTTTATCCTTATCACCCACA K S S S A A S L T S S V S A L S L S P T I 4
0
+421 TCAGCCATAAATATTAGCTCCAAAAGTTTGAGCCCAAAGTTCTCTCATCATAGTAACAGC S A I N I S S K S L S P K F S H H S N S l 6
0
+481 AATACTGCTATTACACCCGCGCCTACTCCCACTGCTTCAAATATTAATAATGTAAATAAG N T A I T P A P T P T A S N I N N V N K I 8
0
+541 ATAACCAATACAAGTGCACCTATTTGTGGGAGGTTTCTTGTGCATAAAGATGGTACCCAT I T N T S A P I C G R F L V H K D G T H 2 0
0
+601 GAACATCACTTAAAAAATGCTAAGAGACAAGAAAAGCTAAGCACAATGATTAAAAACATG E H H L K N A K R Q E K L S T M I K N M 2 2
0
BamHI
+661 GTTGGTGCGAGCAAATTACGTGGTGAGGCAAAATCTGCTGTCCC~GATATAATAAT~ V G A S K L R G E A K S A V P D I I M D 2 4 0 Figure I . The nucleotide and amino acid sequences of a new putative protein kinase located on chromosome 111 from Saccharomyces cerevisiae. Nucleotides and deduced amino acids are numbered on the left and on the right margins, respectively. Putative TATA box, CAAT box, termination and polyadenylation sites are underlined and indicated. The consensus sequences invariant among known protein kinases are indicated by asterisks. The consensus sequences characteristic for serine/threonine kinases are emphasized by white dots. The restriction sites used in the disruption experiment are underlined twice and indicated.
OPEN READING FRAME YCRlOl FROM SACCHAROMYCES CEREVSIAE IS A PUTATIVE PROTEIN KINASE
653
+721 ~AAAGACGACTTTAAAATCCAACAAGAATCCTCCTACTCTTTTTGCAGGCTTCATGAAG P K T T L K S N K N P P T L F A G F M K 2 6
0
+781 CAGGTCGTGGATATGGATGATAAATATCCAGkAGGCGCGCTCCCACAAGTGGCGCTTTAAAT Q V V D M D D K Y P E G A P T S G A L N 2 8
0
+841 TGTCCTGAAAGGGATATATACAGGTCAGATCAAAAAGATTCCAAAAATAATACGCATAAT C P E R D I Y R S D Q K D S K N N T H N 3 0
0
+901 ATCACTACTACTAAAAAAGATAGGCAATGTTTTGCCGAAAAGTATGGTCGCTGTCAAGAA I T T T K K D R Q C F A E K Y G R C Q E 3 2
0
+961 GTCCTTGGTAAAGGTGCTTTTGGTGTAGTAAGAATATGTCAAAAGAAAAATGTTTCTTCT V L G K G A F G V V R I C Q K K N V S S 3 4
0
*
*
* H i ndI II
+I021 CAAGATGGTAATAAAAGTGAAAAGCTTTATGCAGTGAAAGAGTTC~GCGTAGAACATCC Q D G N K S E K L Y A V K E F K R R T S 3 6
0
+lo81 GAATCAGCAGAAAAGTATTCTAAGAGGTTGACTTCTGAATTTTGCATTTCTTCTTCATTA E S A E K Y S K R L T S E F C I S S S L 3 8
0
+1141 CACCATACAAATATTGTTACTACACTAGATCTTTTCCAAGATGCCAAAGGCGAGTACTGT H H T N I V T T L D L F Q D A K G E Y C 4 0
0
+I201 GAAGTAATGGAATATTGTGCAGGTGGCGATCTATTCACTTTGGTCGTTGCCGCCGGAAAA E V M E Y C A G G D L F T L V V A A G K 4 2
0
+1261 TTAGAATATATGGAAGCAGATTGTTTCTTCAAGCAGCTTATTAGAGGTGTTGTTTATATG L E Y M E A D C F F K Q L I R G V V Y M 4 4
0
*
*
*
+1321 CATGAAATGGGTGTTTGTCATAGAGATTTGAAGCCTGAGAACTTACTGCTTACGCACGAT H E M G V C H R D o L o K o P o E o N o L L L T H D 460
* * *
+I381 GGTGTGCTAAAAATTACAGACTTTGGTAACAGCGAATGTTTCAAGATGGCATGGGAAAAA G V L K I T D F G N S E C F K M A W E K 4 8
0
+I441 AATATTCACCTTAGTGGAGGCGTTTGCGGTTCATCGCCGTACATCGCCCCAGAGGAATAT N I H L S G G V C G S S P Y I A P E E Y 5 0
0
+I501 ATCAAAGAAGAGTTTGATCCAAGACCCGTAGATATATGGGCATGTGGTGTCATTTATATG I K E E F D P R P V D I W A C G V I Y M 5 2
0
+I561 GCAATGAGAACTGGTAGACAATTGTGGAGTTCTGCTGAAAAAGACGATCCATTTTATATG A M R T G R Q L W S S A E K D D P F Y M 5 4
0
+I621 AATTATTTAAAAGGACGTAAGGAAAAGGGAGGCTATGAGCCAATCGAAAGTTTAAAAAGA N Y L K G R K E K G G Y E P I E S L K R 5 6
0
+I681 GCCAGGTGTAGGAATGTTATATATTCGATGTTAGATCCCGTTCCGTACAGAAGAATTAAC A R C R N V I Y S M L D P V P Y R R I N 5 8
0
+I741 GGGAAACAAATTTTGAACAGTGAATGGGGAAGGGAGATAAAATGCTGCCATAATGGGCGC G K Q I L N S E W G R E I K C C H N G R 6 0
0
* * *
* * *
Figure I . cont'd
654
J. SKALA E T A L
+1801 G C A T T G A A A T A A A C G A G T A C T T C A C T T T C A A A T A T C A C G A T T C G G T A A L K 603
+1861 TTTTTTACTTAATCTAGTACACTAAGGAATGCTTTGCTTTGTTATCCGGCATTCGTATCTTATTC termination site
+1921 CTCGCTTCTATTGTTCTACTTTTATATCCCGTTTGGCTGATTACGGATCACGTTCA~TT
+1981 GGTAAATCCCATTAATTAAAAAAGAATTGTAATTGTAACCTTATTTAA~AAAAATAGTACATA p l y adenylation site
+2041 ACAATAAAAAAAAAAAAGATAATAATGCTTTTTTGAATTTATTGCTAGACATTCTTACGTTTATT Figure I . i o n r ’ d
In the second domain, conserved sequences diagnostic for protein kinases were detected (Figure 1). The region between residues 323 and 498 includes the consensus sequence Asp-Leu-Lys-Pro-Glu-Asn typical for serine/threonine kinases as well as five consensus sequences characteristic for the catalytic part of the kinases (Hanks et al., 1988). namely Gly-X-Gly-X-X-Gly (residues 323-328), Ala-X-LysX-Phe (residues 35 1-355). Arg-Asp-Leu (residues 448-450), Asp-Phe-Gly (residues 467-469) and Ala-Pro-Glu (residues 496-498). The second domain has 39% identity (Figure 2) with the carboxy-terminal part of the yeast NPRl protein kinase (Vandenbol et al., 1990). This protein of 790 amino acid residues involved in the regulation of amino acid permeation also presents a similar bipartite organization. Its amino-terminal moiety is also serine rich and like the YCR 10 I gene product has an undefined structure while the carboxy-terminus is highly alpha-helices structured and contains the
sequences typical for the catalytic regions of all protein kinases. GENE DISRUPTION Gene disruption experiment was performed using the one-step method (Rothstein, 1983). The 1.17 kbp long BgII fragment from the plasmid pFL34 (gift from Francois Lacroute) containing the URA3 gene was inserted into the unique BarnHI site of YCRIOl between the Hind111 sites (Figure 1). The resulting 2369 bp long Hind111 linear DNA fragment was used for transformation of the yeast haploid (W303-1 B MATalpha and W303- 1 A MATa, both are ade2- I trpl-1 leu2-3,112 his3-11,15 ura3-I, gift from R.J. Rothstein) and diploid (issued from the cross of W303- 1 B and W303- 1 A) strains. Randomly selected transformants have been verified by Southern blotting. Disruption in both diploid and haploid strains was achieved; the derivative strains are viable,
YCR101
374-LGKGAFGVVRICQKKNVSSQDGNKSEKLYAVKEFKRR-TSESAEKYSKRL
NPR1
49O-LGAGAGGSVKLAQRISDN--------KIFAVKEFRTKFENESKRDYVKKI
YCR101
TSEFCISSSLHHTNIVTTLDLFQDAKG-----EYCEVMEYCAGGDLFTLVVAAG
NPRl
TSEYCIGTTLNHPNIIETIEIVYENDRILQVMEYCEY------- DLFAIV-MSN
YCR101
KLEYMEADCFFKQLIRGVVYMHEMGVCHRDLKPENLLLTHDGVLKITDFGNSEC
NPRl
KMSYEEICCCFKQILTGVQYLHSIGLAHRDLKLDNCVINEKGIVKLIDFGAAVV
YCR101
FKMAWEKNIHLSGGVCGSSPYIAPEEYIKEEFDPRPVDIWACGVIYM~-576
NPRl
FSYPFSKNLVEASGIVGSDPYLAPEVCIFAKYDPRPVDIWSSAIIFACM-678
I..IIIII.
I I I I I I.. I.
I l l - I I -.I I 11. I.-I. I I I
*
*
IIII
I I l l . . I 1 1 . 1 4. I I I I I * I - .
4. ~ I . I I I I . I I I I
-IIIIIIII.
II
I I..
-
III-.I
I 4 . Ill
-1-
*
I
Figure 2. Sequence alignment of the carboxy-terminal parts from the putative protein kinasr YCRlOl and NPRl kinasr (Vandenbol er 01.. 1990).Venical dashes and points indicate identical amino acid residues and conservative replacements. respectively. Numbers indicate the positions of the amino acids.
OPEN READING FRAME Y C R I O I FROM SACCHAROMYCES CEREVSIAE IS A PUTATIVE PROTEIN KINASE
showing that the gene under study is not an essential one. Growth of the a and alpha haploid disruptants was tested on solid Y P medium (2% yeast extract, Difco; 1 c/c bacto-peptone, Difco; 2% agar) in 54 conditions including different carbon sources (2% glucose, 2 % galactose, 2% ethanol, 2% lactic acid or 2% glycerol) at three pH values ( 3 , 6 6 and 8.5) and three temperatures (16"C, 28°C and 37°C). Resistance to cycloheximide (0.5 and 1.0 x 10-6 g/ml) and ability for growth in the presence of 2 M-sorbitol and different sodium, potassium or ammonium salt concentrations (0.75 M and 1.5 M) were tested in liquid Y P D medium (2% yeast extract, Difco; I G/o bacto-peptone, Difco; 2% glucose). Only one significant difference was observed. In the presence of sodium salts in the medium, the generation time for the strain with the disrupted gene was t w o times longer than that for wild type. This effect was independent of the nature of the anion present and was not observed in the presence of potassium and ammonium salts. N o defect in cell morphology w a s observed under these conditions.
655
Hanks, S.K., Quinn, A.M. and Hunter, T. (1988). The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 42-52.
Henikoff, S. and Cohen. E.H. (1984). Sequences responsible for transcription termination on a gene segment in Sacchuromyces cerevisiae. Mol. Cell. Biol. 4, 15 15-1 5 2 0 .
Mortimer. R.K., Schild, D., Contopoulou, C.R. and Kans, J.A. (1989). Genetic map of Saccharomyces cerevisiae, Edition 10. Yeasf 5, 321-403. Olson, M.V., Dutchik, J.E.. Graham, M.Y ., Brodeur, G.M., Helms, C., Frank. M., MacCollin, M., Scheinman, R. and Frank, T. (1986). Random-clone strategy for genomic restriction mapping in yeast. Proc. Narl. Acad. S C ~USA . 83,7826-7830.
Palzkill, T.G.. Oliver, S.G. and Newlon C.S. (1986). DNA sequence analysis of ARS elements from chromosome Ill of Soc~charomyce.scerevisioe: identification of a new conserved sequence. Nuc. Acids Kes. 14,6247-6264.
Rothstein, R.J. (1983). One-step gene disruption in yeast. Methods in Enzymology 101, 202-2 1 I .
Sanger, F., Nicklen, S. and Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Null. Acad. Sci. USA 74,5463-5467.
ACKNOWLEDGEMENTS We gratefully acknowledge D r John Sgouros (MIPS, Munich, Germany) for help with sequence analysis. This work was supported in part by grants to A. Goffeau from the Services d e la Politique Scientifique et d e la Region Wallone and the Biotechnology Action Programme from the Commission of the European Communities.
REFERENCES Bennetzen, J.H. and Hall, B.D. (1982). Codon selection in yeast. J . B i o l . Chem. 257, 3026-303 1. Garnier. J., Osguthorpe, D.J. and Robson, B. (1978). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J . Mol. Biol. 120, 97-1 20.
Strathem, J.N., Newlon, C.S.. Herskowitz, I. and Hicks, J.B. (1979). Isolation of a circular derivative of yeast chromosome 111: implications for the mechanism of mating type conversion. Cell 18, 309-3 19. Urdaci, M., Dulau, L., Aigle, M. and Crouzet, M. (1990). Sequence of the yeast gene RVS16I located on chromosome 111. Yeast 6, 173-176. Vandenbol, M., Jauniaux, J.C. and Grenson, M. (1990).The Sacchuromyces cerevisiae NPRI gene required for the activity of ammonia-sensitive amino acid permeases encodes a protein kinase homologue. Mol. Gen. Genet 222,393-399.
Yoshikawa, A. and Isono, K. (1990). Chromosome I11 of Saccharomyces cerevisiae: An ordered clone bank, a detailed restriction map and analysis of transcripts suggest the presence of I60 genes. Yeasr 6,383-401. Zaret, K.S. and Sherman, F. (1982). DNA sequence required for efficient transcription termination in yeast. Cell 28.563-573.
0
VOL. 7: 651-655 (1991)
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Yeast Sequencing Reports
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The Open Reading Frame YCRlOl located on Chromosome I11 from Saccharomyces cerevisiae is a Putative Protein Kinase JACEK SKALA*,B ~ N E D I C T EPURNELLE, MARC CROUZET', MICHEL AIGLE+A N D ANDREGOFFEAU
Unit6 de Biochimie Phpsiologique, UniversitP Cutholique de Louvain,B- 1348 Louvain-la-Neuve,Belgium 'Lahoratoire de BiolocqieMolkculaire et de SPquenqage, Universitt de Bordeuux 11, 146 rue L t o Saignut, 33076 Bordeaux-cedex, France
Received 30 May 1991; accepted 4 June 1991
KEY WORDSxhromosome 111; sequencing; gene distruption; kinase.
As part of the European project of sequencing the yeast Saccharomyces cerevisiue genome, we have sequenced the DlOH and J 1 ID fragments (Palzkill et a/., 1986) of the 524-24a strain (Strathem etal., 1979). Sequencing was carried out on both strands by the dideoxy chain-termination method (Sanger et a/.. 1977). The DlOH and J l l D fragments are contiguous; their junction has been verified by sequencing 1500 bp of the overlapping clone lambda CH4Ap17 from the strain AB972 (Olson et al., 1986). The 1809 bp long open reading frame YCRlOl has been identified at the junction of DlOH and J11D. YCRIOl is located between the SUF2 (Mortimer et d., 1989) and the KVS161 genes (Urdaci et al., 1990) o n the right arm of chromosome 111. The DNA and deduced amino acid sequences are given in Figure 1. ANALYSIS OF THE 5'- AND 3'-NON-CODING REGIONS Two CAAT boxes have been found in the 5'-noncoding region at positions -418 to -415 and -21 1 to -208, as well as two TATA-like sequences at positions -387 to -383 and -211 to -208 (Figure 1). In the 3'-non-coding region a polyadenylation site consensus
AATAAA (Zaret and Sherman, 1982) at position +2043 to +2047 and a termination site consensus TTTTATA (Henikoff and Cohen, 1984) at position + 1940 to + 1946 were detected (Figure 1). ANALYSIS OF THE OPEN READING FRAME The predicted protein comprises 603 amino acids and has a molecular weight of 66,665 Da. The value of codon bias index calculated according to Bennetzen and Hall (1982) is 0.084 indicating a low level of expression. The corresponding mRNA has been characterized by Yoshikawa and Isono (1990). who estimated that the transcript had a relative abundance of 2 in a scale from 1 (lowest) to 5 (highest abundance). Determination of secondary structure using the method of Gamier et a/. (1978) suggests the existence of two domains in the predicted protein. The aminoterminal domain (residues 1-258) present largely undefined structures while the carboxy-terminal domain (residues 259-603) is highly structured in alpha-helices. The first domain is rich in serine residues (18.2%) compared to the second one (6.3%) and has no significant homology with any protein contained in the data bank.
Editorial correspondence to: Andre Goffeau TEL: 32/10/47 36 14 FAX: 32/10/47 38 72 'Permanent address: Institute of Microbiology, Wroclaw University, Przybyszewskiego 63, 5 1-148 Wroclaw, Poland.
0749-S03X/9 1/060643-08$05.00 01991 by John Wiley & Sons Ltd
652
J. SKALA E T A L
-480 CAGCTTTTAAAGAGGGAAGAGGAAACGGAGAAGAAAATATACTACTTTATAGAGATATTC CAAT
TATA
-420 GGCAATTTTTTTTAGCAAAAGTTTATTAAACCCTAATATAGACCTTTACTTGGAGTTTCG -360 ATTTTAAGAATTTTTTATCATTTTTTTTTTTTGACGATTTCTGTCGGTTTCGTTATAACC -300 TGTTGTGTTGTTGTTGTTGTTGTTATTGCTGGGTTGTTGTTATTCACTTTAACATTATCG CAAT
-240 AATAAAATTTGGACTTTCAAAGTCCTGTTCAATAAGTTGTACTAAGCCTTTAAGCGTTTT H i ndI I I
TATA
-180 TAAGTTACAAATAAAAACTGGAAAGCTTAAGGTAGGTAGCTTTCTAATATTAGGCTCTCAACCT -120 TTTTTTACTCTCCATTTACCGCTAGATATTAACTAGTAGTAAGTATTGTGACTACTATTC -60 CCTGTAATCAAAAAAAAAGTAATCAGATTTTATTTTATTTCGACATTACCCCTCAAATAT
+ I ATGACTGGTATGAATGATAATAATGCCGCTATTCCTCAGCAAACTCCAAGGAAACATGCG M
T
G
M
N
D
N
N
A
A
I
P
Q
Q
T
P
R
K
H
A
20
+61 CTATCTTCTAAAGTTATGCAACTTTTTAGAAGCGGTTCAAGATCATCTAGGCAGGGAAAG L S S K V M Q L F R S G S R S S R Q G K 40 +I21 GCCTCATCGAATATCCAGCCACCTTCTAATATAAACACAAACGTTCCATCGGCGTCTAAA A S S N I Q P P S N I N T N V P S A S K 60
+I81 TCAGCCAAATTTGGTTTACATACCCCAACCACTGCTACTCCTAGGGTAGTTTCTAATCCT S A K F G L H T P T T A T P R V V S N P
80
+241 TCTAATACTGCAGGTGTGAGTAAACCGGGCATGTATATGCCCGAATATTACCAGTCGGCA S N T A G V S K P G M Y M P E Y Y Q S A I O
O
+301 TCACCATCGCACTCTAGTTCATCCGCATCATTAAACAACCATATTGATATTAACACCTCT S P S H S S S S A S L N N H I D I N T S l 2
0
+361 AAGTCATCATCAGCTGCTTCTTTAACTTCGTCAGTATCAGCTTTATCCTTATCACCCACA K S S S A A S L T S S V S A L S L S P T I 4
0
+421 TCAGCCATAAATATTAGCTCCAAAAGTTTGAGCCCAAAGTTCTCTCATCATAGTAACAGC S A I N I S S K S L S P K F S H H S N S l 6
0
+481 AATACTGCTATTACACCCGCGCCTACTCCCACTGCTTCAAATATTAATAATGTAAATAAG N T A I T P A P T P T A S N I N N V N K I 8
0
+541 ATAACCAATACAAGTGCACCTATTTGTGGGAGGTTTCTTGTGCATAAAGATGGTACCCAT I T N T S A P I C G R F L V H K D G T H 2 0
0
+601 GAACATCACTTAAAAAATGCTAAGAGACAAGAAAAGCTAAGCACAATGATTAAAAACATG E H H L K N A K R Q E K L S T M I K N M 2 2
0
BamHI
+661 GTTGGTGCGAGCAAATTACGTGGTGAGGCAAAATCTGCTGTCCC~GATATAATAAT~ V G A S K L R G E A K S A V P D I I M D 2 4 0 Figure I . The nucleotide and amino acid sequences of a new putative protein kinase located on chromosome 111 from Saccharomyces cerevisiae. Nucleotides and deduced amino acids are numbered on the left and on the right margins, respectively. Putative TATA box, CAAT box, termination and polyadenylation sites are underlined and indicated. The consensus sequences invariant among known protein kinases are indicated by asterisks. The consensus sequences characteristic for serine/threonine kinases are emphasized by white dots. The restriction sites used in the disruption experiment are underlined twice and indicated.
OPEN READING FRAME YCRlOl FROM SACCHAROMYCES CEREVSIAE IS A PUTATIVE PROTEIN KINASE
653
+721 ~AAAGACGACTTTAAAATCCAACAAGAATCCTCCTACTCTTTTTGCAGGCTTCATGAAG P K T T L K S N K N P P T L F A G F M K 2 6
0
+781 CAGGTCGTGGATATGGATGATAAATATCCAGkAGGCGCGCTCCCACAAGTGGCGCTTTAAAT Q V V D M D D K Y P E G A P T S G A L N 2 8
0
+841 TGTCCTGAAAGGGATATATACAGGTCAGATCAAAAAGATTCCAAAAATAATACGCATAAT C P E R D I Y R S D Q K D S K N N T H N 3 0
0
+901 ATCACTACTACTAAAAAAGATAGGCAATGTTTTGCCGAAAAGTATGGTCGCTGTCAAGAA I T T T K K D R Q C F A E K Y G R C Q E 3 2
0
+961 GTCCTTGGTAAAGGTGCTTTTGGTGTAGTAAGAATATGTCAAAAGAAAAATGTTTCTTCT V L G K G A F G V V R I C Q K K N V S S 3 4
0
*
*
* H i ndI II
+I021 CAAGATGGTAATAAAAGTGAAAAGCTTTATGCAGTGAAAGAGTTC~GCGTAGAACATCC Q D G N K S E K L Y A V K E F K R R T S 3 6
0
+lo81 GAATCAGCAGAAAAGTATTCTAAGAGGTTGACTTCTGAATTTTGCATTTCTTCTTCATTA E S A E K Y S K R L T S E F C I S S S L 3 8
0
+1141 CACCATACAAATATTGTTACTACACTAGATCTTTTCCAAGATGCCAAAGGCGAGTACTGT H H T N I V T T L D L F Q D A K G E Y C 4 0
0
+I201 GAAGTAATGGAATATTGTGCAGGTGGCGATCTATTCACTTTGGTCGTTGCCGCCGGAAAA E V M E Y C A G G D L F T L V V A A G K 4 2
0
+1261 TTAGAATATATGGAAGCAGATTGTTTCTTCAAGCAGCTTATTAGAGGTGTTGTTTATATG L E Y M E A D C F F K Q L I R G V V Y M 4 4
0
*
*
*
+1321 CATGAAATGGGTGTTTGTCATAGAGATTTGAAGCCTGAGAACTTACTGCTTACGCACGAT H E M G V C H R D o L o K o P o E o N o L L L T H D 460
* * *
+I381 GGTGTGCTAAAAATTACAGACTTTGGTAACAGCGAATGTTTCAAGATGGCATGGGAAAAA G V L K I T D F G N S E C F K M A W E K 4 8
0
+I441 AATATTCACCTTAGTGGAGGCGTTTGCGGTTCATCGCCGTACATCGCCCCAGAGGAATAT N I H L S G G V C G S S P Y I A P E E Y 5 0
0
+I501 ATCAAAGAAGAGTTTGATCCAAGACCCGTAGATATATGGGCATGTGGTGTCATTTATATG I K E E F D P R P V D I W A C G V I Y M 5 2
0
+I561 GCAATGAGAACTGGTAGACAATTGTGGAGTTCTGCTGAAAAAGACGATCCATTTTATATG A M R T G R Q L W S S A E K D D P F Y M 5 4
0
+I621 AATTATTTAAAAGGACGTAAGGAAAAGGGAGGCTATGAGCCAATCGAAAGTTTAAAAAGA N Y L K G R K E K G G Y E P I E S L K R 5 6
0
+I681 GCCAGGTGTAGGAATGTTATATATTCGATGTTAGATCCCGTTCCGTACAGAAGAATTAAC A R C R N V I Y S M L D P V P Y R R I N 5 8
0
+I741 GGGAAACAAATTTTGAACAGTGAATGGGGAAGGGAGATAAAATGCTGCCATAATGGGCGC G K Q I L N S E W G R E I K C C H N G R 6 0
0
* * *
* * *
Figure I . cont'd
654
J. SKALA E T A L
+1801 G C A T T G A A A T A A A C G A G T A C T T C A C T T T C A A A T A T C A C G A T T C G G T A A L K 603
+1861 TTTTTTACTTAATCTAGTACACTAAGGAATGCTTTGCTTTGTTATCCGGCATTCGTATCTTATTC termination site
+1921 CTCGCTTCTATTGTTCTACTTTTATATCCCGTTTGGCTGATTACGGATCACGTTCA~TT
+1981 GGTAAATCCCATTAATTAAAAAAGAATTGTAATTGTAACCTTATTTAA~AAAAATAGTACATA p l y adenylation site
+2041 ACAATAAAAAAAAAAAAGATAATAATGCTTTTTTGAATTTATTGCTAGACATTCTTACGTTTATT Figure I . i o n r ’ d
In the second domain, conserved sequences diagnostic for protein kinases were detected (Figure 1). The region between residues 323 and 498 includes the consensus sequence Asp-Leu-Lys-Pro-Glu-Asn typical for serine/threonine kinases as well as five consensus sequences characteristic for the catalytic part of the kinases (Hanks et al., 1988). namely Gly-X-Gly-X-X-Gly (residues 323-328), Ala-X-LysX-Phe (residues 35 1-355). Arg-Asp-Leu (residues 448-450), Asp-Phe-Gly (residues 467-469) and Ala-Pro-Glu (residues 496-498). The second domain has 39% identity (Figure 2) with the carboxy-terminal part of the yeast NPRl protein kinase (Vandenbol et al., 1990). This protein of 790 amino acid residues involved in the regulation of amino acid permeation also presents a similar bipartite organization. Its amino-terminal moiety is also serine rich and like the YCR 10 I gene product has an undefined structure while the carboxy-terminus is highly alpha-helices structured and contains the
sequences typical for the catalytic regions of all protein kinases. GENE DISRUPTION Gene disruption experiment was performed using the one-step method (Rothstein, 1983). The 1.17 kbp long BgII fragment from the plasmid pFL34 (gift from Francois Lacroute) containing the URA3 gene was inserted into the unique BarnHI site of YCRIOl between the Hind111 sites (Figure 1). The resulting 2369 bp long Hind111 linear DNA fragment was used for transformation of the yeast haploid (W303-1 B MATalpha and W303- 1 A MATa, both are ade2- I trpl-1 leu2-3,112 his3-11,15 ura3-I, gift from R.J. Rothstein) and diploid (issued from the cross of W303- 1 B and W303- 1 A) strains. Randomly selected transformants have been verified by Southern blotting. Disruption in both diploid and haploid strains was achieved; the derivative strains are viable,
YCR101
374-LGKGAFGVVRICQKKNVSSQDGNKSEKLYAVKEFKRR-TSESAEKYSKRL
NPR1
49O-LGAGAGGSVKLAQRISDN--------KIFAVKEFRTKFENESKRDYVKKI
YCR101
TSEFCISSSLHHTNIVTTLDLFQDAKG-----EYCEVMEYCAGGDLFTLVVAAG
NPRl
TSEYCIGTTLNHPNIIETIEIVYENDRILQVMEYCEY------- DLFAIV-MSN
YCR101
KLEYMEADCFFKQLIRGVVYMHEMGVCHRDLKPENLLLTHDGVLKITDFGNSEC
NPRl
KMSYEEICCCFKQILTGVQYLHSIGLAHRDLKLDNCVINEKGIVKLIDFGAAVV
YCR101
FKMAWEKNIHLSGGVCGSSPYIAPEEYIKEEFDPRPVDIWACGVIYM~-576
NPRl
FSYPFSKNLVEASGIVGSDPYLAPEVCIFAKYDPRPVDIWSSAIIFACM-678
I..IIIII.
I I I I I I.. I.
I l l - I I -.I I 11. I.-I. I I I
*
*
IIII
I I l l . . I 1 1 . 1 4. I I I I I * I - .
4. ~ I . I I I I . I I I I
-IIIIIIII.
II
I I..
-
III-.I
I 4 . Ill
-1-
*
I
Figure 2. Sequence alignment of the carboxy-terminal parts from the putative protein kinasr YCRlOl and NPRl kinasr (Vandenbol er 01.. 1990).Venical dashes and points indicate identical amino acid residues and conservative replacements. respectively. Numbers indicate the positions of the amino acids.
OPEN READING FRAME Y C R I O I FROM SACCHAROMYCES CEREVSIAE IS A PUTATIVE PROTEIN KINASE
showing that the gene under study is not an essential one. Growth of the a and alpha haploid disruptants was tested on solid Y P medium (2% yeast extract, Difco; 1 c/c bacto-peptone, Difco; 2% agar) in 54 conditions including different carbon sources (2% glucose, 2 % galactose, 2% ethanol, 2% lactic acid or 2% glycerol) at three pH values ( 3 , 6 6 and 8.5) and three temperatures (16"C, 28°C and 37°C). Resistance to cycloheximide (0.5 and 1.0 x 10-6 g/ml) and ability for growth in the presence of 2 M-sorbitol and different sodium, potassium or ammonium salt concentrations (0.75 M and 1.5 M) were tested in liquid Y P D medium (2% yeast extract, Difco; I G/o bacto-peptone, Difco; 2% glucose). Only one significant difference was observed. In the presence of sodium salts in the medium, the generation time for the strain with the disrupted gene was t w o times longer than that for wild type. This effect was independent of the nature of the anion present and was not observed in the presence of potassium and ammonium salts. N o defect in cell morphology w a s observed under these conditions.
655
Hanks, S.K., Quinn, A.M. and Hunter, T. (1988). The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 42-52.
Henikoff, S. and Cohen. E.H. (1984). Sequences responsible for transcription termination on a gene segment in Sacchuromyces cerevisiae. Mol. Cell. Biol. 4, 15 15-1 5 2 0 .
Mortimer. R.K., Schild, D., Contopoulou, C.R. and Kans, J.A. (1989). Genetic map of Saccharomyces cerevisiae, Edition 10. Yeasf 5, 321-403. Olson, M.V., Dutchik, J.E.. Graham, M.Y ., Brodeur, G.M., Helms, C., Frank. M., MacCollin, M., Scheinman, R. and Frank, T. (1986). Random-clone strategy for genomic restriction mapping in yeast. Proc. Narl. Acad. S C ~USA . 83,7826-7830.
Palzkill, T.G.. Oliver, S.G. and Newlon C.S. (1986). DNA sequence analysis of ARS elements from chromosome Ill of Soc~charomyce.scerevisioe: identification of a new conserved sequence. Nuc. Acids Kes. 14,6247-6264.
Rothstein, R.J. (1983). One-step gene disruption in yeast. Methods in Enzymology 101, 202-2 1 I .
Sanger, F., Nicklen, S. and Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Null. Acad. Sci. USA 74,5463-5467.
ACKNOWLEDGEMENTS We gratefully acknowledge D r John Sgouros (MIPS, Munich, Germany) for help with sequence analysis. This work was supported in part by grants to A. Goffeau from the Services d e la Politique Scientifique et d e la Region Wallone and the Biotechnology Action Programme from the Commission of the European Communities.
REFERENCES Bennetzen, J.H. and Hall, B.D. (1982). Codon selection in yeast. J . B i o l . Chem. 257, 3026-303 1. Garnier. J., Osguthorpe, D.J. and Robson, B. (1978). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J . Mol. Biol. 120, 97-1 20.
Strathem, J.N., Newlon, C.S.. Herskowitz, I. and Hicks, J.B. (1979). Isolation of a circular derivative of yeast chromosome 111: implications for the mechanism of mating type conversion. Cell 18, 309-3 19. Urdaci, M., Dulau, L., Aigle, M. and Crouzet, M. (1990). Sequence of the yeast gene RVS16I located on chromosome 111. Yeast 6, 173-176. Vandenbol, M., Jauniaux, J.C. and Grenson, M. (1990).The Sacchuromyces cerevisiae NPRI gene required for the activity of ammonia-sensitive amino acid permeases encodes a protein kinase homologue. Mol. Gen. Genet 222,393-399.
Yoshikawa, A. and Isono, K. (1990). Chromosome I11 of Saccharomyces cerevisiae: An ordered clone bank, a detailed restriction map and analysis of transcripts suggest the presence of I60 genes. Yeasr 6,383-401. Zaret, K.S. and Sherman, F. (1982). DNA sequence required for efficient transcription termination in yeast. Cell 28.563-573.
