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The Complete Sequence of a 6146 bp Fragment of Saccharomyces cerevisiae Chromosome I11 Contains Two New Open-Reading Frames CATHAL WILSON, PAOLA GRISANTI AND LAURA FRONTALI* Department of Cellular and Developmental Biology, University of Rome 'La Sapienza', Piazzale Aldo Moro 5 , 00185 Rome, Italy
Received 30 January 1992; accepted 5 February 1992
As part of the EEC project to sequence the entire chromosome I11 of Saccharomyces cerevisiae we have sequenced a total of 1 1,040 bp from near the right end of the chromosome. A new protein kinase gene was found at one extremity of the sequenced region (Wilson et al., 1992), while the previously sequenced actin binding protein gene, ABPI, (Drubin et al., 1990) was found at the other extremity. We present here the sequence of the region between these two genes which has the potential to code for two new open reading frames (ORFs).
MATERIALS AND METHODS
DNA manipulations and sequencing strategy
Cloning, restriction analysis and preparation of plasmid DNA were essentially as described in Maniatis et al. (1982). A restriction map was constructed for the entire PM3712 insert and restriction fragments covering the left half of the insert were subcloned in both orientations in pTZl8R/pTZ19R. Directed overlapping deletions were generated using DNaseI (Lin et al., 1985) or exonuclease I11 (Henikoff, 1984). Sequencing of remaining gaps and across subclone Strains and plasmids boundaries was performed using oligonucleotideThe Escherichia coli strains used were JM101, directed sequencing of double-stranded DNA. Both JM109 and JM83 (Yannisch-Perron et al., 1989). double-stranded and single-stranded DNA were The S. cerevisiae strain W303-1B (MATa,ura,trp, used for sequencing using the chain-termination his,leu,ade) and its isogenic a and diploid derivatives method of Sanger et al. (1977). (R. Rothstein) were used for the gene disruption experiments. Subcloning, double-stranded DNA manipu- Gene disruption lations and production of single-stranded DNA The one-step gene-disruption technique was used were performed using the vectors pTZ18R/pTZ19R (Rothstein, 1983). A 489 bp SpeI fragment located (Pharmacia). The plasmid pFL39HIS' contains a between nucleotides 2062 and 2551 (Figure 1) was 1.76 kb insert carrying the yeast HIS3 gene and was replaced by the 1.76 kb fragment from pFL39HIS'. kindly provided by Professor P. Slonimski. Transformation of intact yeast cells was as described *Addressee for correspondence. by Ausubel et al. (1988) except that LiCl was used Chromosome 111DNA The lambda clone PM3712 (constructed by Professor M. Olson) contains an insert of 2&21 kb. Preparations of PM37 12 DNA were provided by Professor S. Oliver and Dr M. Gent. The insert DNA comes from the right end of chromosome I11 and is situated between the ABPI and rosl (cdc39) genes (Mortimer et al., 1989).
0749-503X/92/070569-07 $08.50 0 1992 by John Wiley & Sons Ltd
570
C. WILSON ETAL. -491
C C C m C A C T ~ T C M C G C C T P ~ C T C T T ~ C M ~ A T A T G A C T C C T C T A C A C T A ~ A ~-400 C M ~ G
-401
-312 A C C C I C T G C C C ~ A C C C C A ~ ~ ~ C C T C G M C A T ~ C M C A C T ~ ~ ~ A ~ ~ C T C C T ~ T C ~ T A C A ~ T ~ ~
-311
T A T C T T A C T A C M C C A T M T C C T C m T A C M T C C C A T C G C A T C A ~ ~ A C C A ~ C T C T A T C C I A U T C C I G C m C C ~ A T G C T C A -222 ~A~
-22 I
T C G G A m A C ~ C T A C T C T A T C T A T A G A ~ ~ A G A ~ C C A ~ M ~ A ~ T G G M C G ~ M G A ~ M G ~ T ~ A-132 T G M G A G T C
-131
T C C C A C T A G A T A T A T A M C C T C M T T A M T T C A C T A C M T C C M G
-42
-4 I
C A C A C T A C M A M T A C C C T A T C A T C T A C C G M T A T M C T C C M T YCR1102 M N S F A S L G L I Y S V V N L
48 16
49 17
138 ~MCTACACTACACCCTCAMTTCTCTACCAGMTACTACTAC~CACT~CT~CCCTA~A~ATCCACCTCMTAGC A~T L T R V E A Q I V F Y Q N S S T S L P V P T L V S T S I A D 46
139 47
m C A C G A G T C C T C A T C M C T C G C C M C T ~ ~ A ~ C A T C C 228 T C 76 F H E S S S T G E V Q Y S S S Y S Y V Q P S I D S F T S S S
219 77
~ T C T T M C M ~ C M C C T CAC TC C C A M C T T C T T C C A G C T A T G C F L T S F E A P T E T S S S Y A V S S S L I T S D T F S S Y
319 107
408 TCECATATCTPCCATCMCAMCMCTTCATTMTATCMCCTCA~MTATCMCCTCA~GCCT~TC~GAM~CTC~CCACC~C~CMCT~A S D I F D E E T S S L I S T S A A S S E K A S S T L S S T A 136
409 137
C M C C T C A T A G G A C A T C T C A C T C T T C C T C T T C A T T C C A C C T G
499 167
A C A T T T A C C T C A G l T M T C C A T C ~ ~ ~ A ~ C A T T T M C T C A ~ T C T A G C ~ C m C C T C M C ~ T588 A G A ~ A ~ ~ 196 T F T S V N P S Q S W T S F N S E K S S A L S S T I D F T S
589 197
T C T C A G A ~ C A C C T T C M C A T C T C ~ G A G C C T ~ A M ~ C ~ T A C ~ C C C C I A ~ A T M ~ C A T T C T T A ~ C T678 C~CTC~CT 226 S E I S C S T S P K S L E S F D T T C T I T S S Y S P S P S
679 227
TCAAAAMTTCTMCCAGACCTCACTACTCAGCCCA~AGCCTCT~CCA~TCTTCA~AGAmMTA~~~CMCTA ~CM 768
769 257
GCTACTACCMTCACCAMClT~CTA~CCMCTTCTT~GACGCCACATCCTCA~ACCACCMCA~~~CATCCACT ATG 858 286 A T T N D Q T S K T I P T L V D A T S S L P P T L R S S S M
859 287
C C A C C M ~ C T C m C T C A ~ C C T C A C A C M ~ A C G A G C C C C C C C T C T ~ C M C T C C I M C T A C ~ T948 ~ G A ~ ~ C
949 317
T C M T A G A T C T C T T C T C T A T T T A C M C T A C G A ~ G M T A l T C A T C T S I D P S L F T T T S E Y S S T Q L S S L N R A S K S E T V
Q P H R T S H S S S S F E L P V T A P S S S S L P S S T S L
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318 106
498 166
256
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1039 347
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1309 437
A C C A C A T C C C C C C C F T A T C m C T A ~ C A C ~ C C C T T G A C ~ G A T C A C C ~ T A T ~ A C A T C C I C T C C T C T M C A1398 ~CC T T S P A Y V S T A T K T V D G V I T E Y V T W C P L T Q T 466
1399 467
h M T C C ~ C C A ~ C T A G C ~ C C A C M G C C T C C T C A T T T A G C C C I A ~ C T A ~ ~ C 1488 C M ~ C C 496 K S Q A I G V S S S I S S V P Q A S S P S G S S I L S S N S
1489 497
A C C A C T T C T T C C T C C C T C C M C M C ~ C ~ M T C M C T C C M G C G1578 526 S T L A A S N N V P E S T A S C S S Q Y Q D U S S S S L P L
1579 527
T C A ~ C C A C T T G C C T T C T C A T C M C A C M C P M T A C A C M ~ C T C T M C C T C M C C A C A T C C C C ~ A T ~ C T A C ~ ~1668 CC 556 S Q T T W V V I N T T N T Q C S V T S T T S P A Y V S T A T
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Figure 1.
M 1218 C
406
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SEQUENCE OF 6146 bp FRAGMENT OF S. CEREVISIAE CHROMOSOME 111 1669 557
A A M C G G T T G A C C G G G T G A T C C C C M T A T C T T A T ~ A ~ T ~ T G T C C T ~ M ~ ~ C ~ T C A C M ~ ~ ~ T1758 ~~CATCCACG 586 K T V D G V I T E Y V T U C P L T Q T K S Q A I G I S S S T
1759 587
A T T A C C G C C A C A C A M C C T C T A M C ~ T C M T A T T M C A T C T 1848 616 I S A T Q T S K P S S I L T L C I S T L Q L S D A T F K G T
1849 617
G A A A C T A T A A A C A C C C A T C T C A T ~ C C G A M G T A C T T C A G C 1938 646 E T I N T H L M T E S T S I T E P T Y P S G T S D S F Y L C
1939 647
A C C A G T G M G T T M T C T T G C G T C T T C C T T A T C T T C T T A T C C A M ~ C A T C ~ C A G M ~ C T C T A C ~ C ~ C C A T T A C T M C T ~ 2028 ACC 676 T S E V N L A S S L S S Y P N F S S S E G S T A T I T N S T
2029 677
C T T A C A T T T G G A T C G A C C A C M G T A T C C A T C T A C T A G T C T C A
2119 707
C T C A C T G A T m A C T T C A T T C M C C G A A A C C A T C G C A G ~ A T A T C T M T A T T C A C ~ C T T C G T C A M T A G A C T A T T ~ ~ G A C G 2208 L T D F T S N S T E T I A V I S N I H K T S S N K D Y S L T 736
2209 737
A C T A C G C A A T T A A A G A C C A G C G G A M C C A M C G C T T G T G C T T C G
2299 767
T C C T G C C C G G C A T C M G T A ~ G C ~ A T A C G A C A T C C A T A T W2388 796 W C P A S S I A Y T T S I S Y K T L V L T T E V C S H S E C
2389 797
ACTCCMCGGTTATTACWGTG~ACTGCMCMGCTCTACMTCCCCC~ATCMCCTCTAGCTCTAC~TATTATCTTCTACAGTA 2478 T P T V I T S V T A T S S T I P L L S T S S S T V L S S T V 826
2479 827
TCCGMGGTGCAAAAAATCCCCCTGCTTCTGMGTMCTATTMTACCCMGmCTGCTACTTCCGMGCTACTACTACTAGCACTCM S E G A K N P A A S E V T I N T Q V S A T S E A T S T S T Q
2569 857
G T G T C T G C T A C T T C T C C G A C G G C C A C T G C T A ~ G A C A C T T 2658 V S A T S A T A T A S E S S T T S Q V S T A S E T I S T L G 886
2659 887
A C T C A A A A C T T T A C C A C T A C T G G A A G C T T A C m T C C C G G C m A
2749 917
A T T A T T A G T A C A G A C G T A T G T T C C C A T T C C A A A T G T C
2839 947
CATTCTTCTWLAACTCTACACGGAGGCAGTAGACGTGACATTGTCATCCCAT~CCGTMCTATGAGTACC~GTATGTTCTMT 2928 976 H S S Q T L Q T E A V E V T L S S H Q T V T M S T E V C S N
2929 977
TCGAmGCACACCGACTGTTATTACATCTGTGWLAATGAGMGTACTCC~CCATACTTMC~CTTCMCGTCMGTTCCTC~TA 3018 S I C T P T V I T S V Q M R S T P F P Y L T S S T S S S S L 1006
3019 1007
G C C T C C A C C A A A A A M C T T C C T T A ~ G C T C C T C A G A A A T G T 3108 A S T K K S S L E A S S E M S T P S V S T Q S L P L A P T C 1036
3109 1037
T C A G A A A A A C G C T C C A C C A C A T C T G T C T C T C M T G G T C A A A T C A
3199 1067
ACAAATGAAAAGCCCAGTAGACTACCTCTCCATACMCTTCTC~C~TACTCmACCTTCTTC~CTACACC~CCCAATATTCA3288 T N E K P S S T T S P Y N P S S G Y S L P S S S T P S Q Y S 1096
3289 1097
C T A T C T A C A G C T A C T A C M C M T C M C ~ M T C A A M C T G T G T A ~ C M C ~ ~ G T C C A T T G G C A ~ T C T A C T G T A G C T G C T T3378 CT L S T A T T T I N C I K T V Y T T U C P L A E K S T V A A S 1126
3379 1127
TCTCMTCTTCCCCCAGTCTGACACGmcmCGTCGTCGTCAAAACCATCCTCATCT~ATCTCAGACCTCTA~CMTATACA~ATCT 3468 S Q S S R S V D R P V S S S K P S S S L S Q T S I Q Y T L S 1156
3469 1157
ACTGCTACCACCACCATMGTGGTTTGAAW\CTGTATACACGACTTGGTGTCCA~MCM~AAATC~CTTTA~GCTACTACTCM 3558 T A T T T I S G L K T V Y T T W C P L T S K S T L G A T T Q 1186
3559 1187
A C T T C C T C G A C A G C C A M G T T A G A A T T A C T T C C G C T T C A T C T G C M C A T C T A C ~ C T A ~ C ~ ~ ~ C ~ ~ C A ~ T3648 CA~TCT T S S T A K V R I T S A S S A T S T S I S L S T S T E S E S 1216
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Figure 1. Continued.
2118 706
2298 766
2568 856
2748 916 2838 946
3198 1066
572
C . WILSON ET AL. 3649 1217
T C A T C T G C A T A ~ G T C C ~ C A ~ A ~ ~ T ~ C ~ ~ T G T ~ C M ~ C M T C A T C 3738 ~ C A C C T ~ C M C ~ A 1246 S S G Y L S K G V C S C T E C T Q D V P T Q S S S P A S T L
3739 1247
3828 GCATATTCCCCCTCT~CTACATCATCATCATCATCA~CTCMCMCMCT~TCMCA~MCGTCMCACACACCTCTGTCCCG A Y S P S V S T S S S S S P S T T T A S T L T S T H T S V P 1276
3829 1277
T T A ~ A C C A T C A T C T A ~ ~ A T A T C A ~ T C ~ ~ C C A T C A T C M C T T C ~ ~ A T C C A ~ ~ A C C A T C T C C3918 C~ACGTCA L L P S S S S I S A S S P S S T S L L S T S L P S P A P T S 1306
3919 1307
TCMCACTTCCMCAGCMCAGCA G T A T ~ C ~ C C A C m C A T A S T L P T A T A V S S S T P I I S S L P L S S K S S L S L S
4009 1337
C C A G T C T C G T C G T C T A T m C G T C T C A ~ C A T C A T C A T C A T C A T C A T C A T ~ T C A T T ~ T C ~ ~ C A T C T ~ C T A T6098 ATCA P V S S S I L ~ S Q I S S S S S S S S S L A S L P S L S I S 1366
4099 1367
C C M C T ~ G A C A C T T G m ~ C C M C C M C T A ~ C C A T C G C M C A C T M ~ ~ C A G A C T C A C M T ~ C M C A G C A4188 ~ATCC P T V D T V S V L Q P T T S I A T L T C T D S Q C Q Q E V S 1396
4189 1397
A C T A T C T C T M T G G A T C C G A C G A T G T G A C T T C M C T C T I C N C S N C D D V T S T A T T P P S T V T D T U T C T G
4279 1427
T C T G A C T C C C A ~ C C A ~ T C T A ~ ~ G T G A T ~ A C T C G T ~ ~ G T A T C C ~ C G T A T A M T C M4368 ~~A~TATCT~ S E C Q K T T S S S C D G Y S C K V S E T Y K S S A T I S A 1456
4369 1457
TGTAGTGGAGMGGATGCCCC~ACMGTGA~AMTTCTCMTACGTCACGATGACGTCTGTCATTACCCCMGTGCCATA 4458 C S G E G C Q A S A T S E L W S Q Y V T ~ T S V I T P S A I 1486
4459 1487
ACMCMChTCACTGGMCTGCATTCMCTGMTCCACTATATCMTTACTA~GT~~CA~ACATATACATCCAGT~TACTMT 4548 T T T S V E V B S T E S T I S I T T V K P V T Y T S S D T N 1516
4549 1517
G G A W V \ C T G A T M C C A T M C A A ~ C C A C C ~ C T ~ M T T C C A T C A G T M C G A C ~ T M T M C G A G M C ~ G T f f i C C4638 ATM~~
4639 1547
G C A C C A M G C W V L C M C T A C G A C C I A T G T C G A ~ C G A ~ C C T C C A G T ~ A T T G C T A C T T ~ ~ ~ ~ G C A T C 4728 CT~CT
4729 1577
T G G A T T A ~ A C A C C C A T T G T C A G T A C G T A T G C T ~ C ~ G T ~ ~ C T ~ G T A G T M ~ ~ A T G A T M T 4818 ~MT~GATC 1606 U I T T P I V S T Y A G S A S K P L C S K P P U I U V U V I
4819 1607
M ~ C A ~ M ~ C C C G T A T A T A T T A T A T G T A C G T A T A T A T ~ A T A M C A ~ C A C T ~ A C T T A C C ~ M T M4908 T~~TGA N P I * * K G K I I S S S 1I5
G E L I T I T S S S Q T V I P S V T T I I T R T K V A I T S
A P K P T T T T Y V E Q R L S S S G I A T S P V A A A S S T
C C 4008 1336
4278 1426
1546
1576
4998 ~CA~A~CCCC~ 4909 ~ T C A G T C A ~ ~ A C T T C T T C C C C M ~ G T C A T C A T M T C A T A C C A ~ C A ~ A T C ~ ~ A ~ G G C A T P D T H T V E E G L N D D Y D Y W E N D E L Q C S U V K G S 145 174
T C M ~ ~ ~ T ~ ~ T C 4999 A C T T A G A G A M C T T C A A A C G ~ G T C ~ T ~ ~ ~ ~ M ~ C A C M C C T C T G C M 5088 S L S V E P T I T D P Y P K I L E C G R C D L S L P I P S D 115 144 CATCCTC 5089 T T ~ C A C C T T C G A T A M C C A T G ~ ~ C C ~ C A T ~ A T C T T ~ C C A ~ G T C G T C A C T C T G A T ~ ~ A M T A T T 5178 E K V K S L G H K K R V D K I K A W K D D S Q D T L Y E D E a5 114 5179 84
A A A A C C G C A C A C C T I M C T G A ~ T T ~ G M C ~ ~ A C A C T T C A T ~ ~ G A T ~ ~ A C ~ G A T G C C G G C A T5268 AGCATA 55 P A C L N V S L E K S C P K C K U L P S A T G K S A P U A Y
5269
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54
E E P T N I I V K S D H L E R C S T C Q L D P A P E A R S E
5358 25
5359 24
G m G T A T T ~ C M T T G A A A C C f ~ T C A C ~ ~ G A T M T G 5448 1 T G N T N E I S V K T V R E S L T A K L V L P L P U YCR1104
5449
m ~ A T T ~ M ~ ~ C m ~ ~ C A T T A ~ ~ A T A M ~ ~ C T T G A C5538 G C T T ~ M ~ C A T
5539
T A C M G C M T G G A M C M M T T M G A l l M T G T A C G C M C W U G M C T A M
5629
CTAAATTATCTTATACTAGCMASCTT
5628
5655
Figure 1. Continued. Nucleotide and amino acid sequence ofYCRl102 and YCRl104. The stop codon TAA is represented by *. YCRl102 is coded for by the top strand while YCRl104 is coded for by the bottom strand.
SEQUENCE OF 6146 bp FRAGMENT OF S. CEREVISIAECHROMOSOMEI11
and boiled sonicated salmon sperm DNA was used as a carrier (Schiestl and Gietz, 1989). Total DNA was prepared from yeast cells as described by Ausubel et al. (1988).
573
similar to the conserved sequence elements GTAPyGT . . . . TACTAAC . . . . PyAG, which are necessary for the splicing of yeast introns (Woolford, 1989). However, the presence of a T instead of the invariable G (which is important for the correct splicing of the intron) at position 5 of the RESULTS AND DISCUSSION 5' element and the fact that such a splicing event Sequence analysis would disrupt the O R F makes it unlikely that it is an A comparison of our sequenced region with the intron. The YCRllO2 OR F could code for a large proGenEMBL database showed that the left-most part tein of 1609 amino acids. No significant homologies contained the actin binding protein gene A B P l were found with any proteins in the NBRF data(Drubin et al., 1990) beginning at amino acid base. The protein is rich in serine (26.3%) and number 15 of the ORF (data not shown). We found 12 differences between our sequence and the pub- threonine (18.1 %) residues. It also contains a large lished sequence (Table I), which are probably due to number of N-glycosylation sites (see below) which, strain polymorphism, as has been noted previously together with the possibility of 0-glycosylation on serine residues, may mean that the protein is in similar comparisons (e.g. Rad et al., 1991). heavily glycosylated. A hydrophobicity plot of the YCRl102 protein using the algorithm of Kyte and Table 1. Sequence changes in the ABPl gene Doolittle did not reveal any possible membranespanning regions. However, there is a hydrophobic N-terminal region and a very hydrophobic carboxy Nucleotide Previous New Amino acid tail (data not shown). position* sequence sequence change One of the most notable features of the YCRl102 ORF is the presence of a number of repeated 1674 C T S-rL regions, the most striking being an 81 amino acid 1990 T C almost-perfect repeat in the region between amino T 2436 A I+K acid residues 396 and 585 (repeat I . 1 and 1.2, Figure 3134 C T 2). Only three nucleotide changes (and one amino 3298 T C 3' acid change) occur in these repeats. Two other 3442 T C 3' repeated regions are also shown in Figure 2. Repeat 1921-1923 CCG TTC 3' 2.1 is in fact part of repeat 1 . 1 and 1.2. Thus this 19261928 TTC CAG 3' region is represented five times in the ORF. Figure 3 shows the locations of the repeated *Position numbers refer to the data of Drubin et nl. (1990). 3' regions, the 17 N-glycosylation sites and the 26 refers to the non-coding3' end of the sequence. cysteine residues in the YCRllO2 ORF. Of the 26 cysteine residues, 11 are accounted for by the The region between the A B P l gene and the repeated regions. Of the remaining 15 cysteines, ten KIN82 protein kinase gene (Wilson el al., 1992) are located in a short region of 77 amino acids. contains two ORFs, YCRl102 and YCRllO4, on These ten cysteines are arranged in five pairs with opposite strands, whose sequences are shown in four amino acid residues between the two cysteines Figure 1. The smaller of the ORFs, YCR1104, is of each pair. These cysteine residues might be preceded by a TATA box and a poly(dA-dT)-rich involved in intra- or intermolecular disulphide bond sequence which may act as a promoter element for formation and have a role in the stability of the constitutive transcription in yeast (Struhl, 1985). protein product of YCRl102. The presence of the The O RF could code for a protein of 182 amino majority of the N-glycosylation sites in the amino acids which has no remarkable features and which half of the protein and the majority of the cysteine did not reveal significant homology to any proteins residues in the carboxy half may represent two when compared with the NBRF database. different domains of the protein. One possibility is The upstream region of YCRl102 also contains that YCRllO2 codes for a cell wall protein. Cell wall a number of TATA boxes. Within the OR F the proteins are glycosylated, contain disulphide bonds sequence GTACTT . . . . TACTAAC . . . . CAG and some are rich in serine and threonine (Frevert (nucleotide positions 1880-2048 in Figure 1) is and Ballou, 1985).
574
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Figure 2. Repeated regions in the YCRl102 open reading frame. Both the nucleotide and amino acid sequences areshownforrepeats 1.1 and 1.2.A blackdotsignifiesanucleotidechange. Repeat2.1 isapart ofrepeats 1.1 and 1.2.
REPEAT
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6 6
Figure 3. Features of the YCR 1102 open reading frame. The repeated regions are shown by open boxes and the circled numbers. The locations of the cysteine residues are marked with a r and the N-glycosylation sites by A .
Gene disruption of YCR1102 The YCRllO2 gene was disrupted by replacing an internal 489 bp SpeI fragment with the HIS3 gene using the one-step gene-disruption method (Rothstein, 1983). Gene disruption was verified by Southern analysis (data not shown). No differences were found between the disrupted and nondisrupted cells when tested for growth rate,
mating, sporulation, spore viability, mitochondria1 stability, growth on different carbon sources, growth on high concentrations of NaC1, high and low temperature sensitivity and resistance to zymolyase or Kluyveromyces lactis killer toxin. Reduced stringency hybridization suggested that YCR1102 is present in a single copy in the S. cerevisiae genome (data not shown).
SEQUENCE OF 6146 bp FRAGMENT OF S. CEREMSIAE CHROMOSOME 111
ACKNOWLEDGEMENTS This work was supported by the C.N.R. and by Istituto Pasteur-Fondazione Cenci Bolognetti. C.W. was supported by Commission of the European Communities grant number BAP-0553-1. We thank the staff at MIPS for help with databank comparisons. REFERENCES Ausubel, F., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. (Eds) (1988). Current Protocols in Molecular Biology, vol. 2. John Wiley, New York. Drubin, D. G., Mulholland, J., Zhu, Z. and Botstein, D. (1990). Homology of a yeast actin-binding protein to signal transduction proteins and myosin-1. Nature 343, 288-290. Frevert, J. and Ballou, C. E. (1985). Saccharomyces cerevisiae structural cell wall mannoproteins. Biochemistry 24,753-759. Henikoff, S . (1984). Unidirectional digestion with exonuclease I11 creates targeted breakpoints for DNA sequencing. Gene 28,351-359. Lin, H., Lei, S. and Wilcox, G. (1985). An improved DNA sequencing strategy. Anal. Biochem. 147,114-1 19. Maniatis, T., Fritsch, E. F. and Sambrook, J. (Eds) (1982). Molecular Cloning. Cold Spring Harbor Laboratories, New York.
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Mortimer, R. K., Schild, D., Contopoulou, C. R. and Kans, J. A. (1989). Genetic and physical maps of Saccharomyces cerevisiae, edition 10. Yeast 5,321-403. Rad, M. R., Lutzenkirchen, K., Xu, G., Kleinhans, U. and Hollenberg, C. P. (1991). The complete sequence of a 11,953bp fragment from C l G on chromosome 111 encompasses four new open reading frames. Yeast 7, 533-538. Rothstein, R. (1983). Methods in Enzymology 101, 202-2 11. Sanger, F., Nicklen, S. and Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463-5467. Schiestl, R. H. and Gietz, R. D. (1989). High efficiency transformation of intact yeast cells using singlestranded nucleic acids as a carrier. Curr. Genet. 16, 339-346. Struhl, K. (1985). Naturally occurring poly(dA:dT) sequences are upstream promotor elements for constitutive transcription in yeast. Proc. Natl. Acad. Sci. USA 82,8419-8423. Wilson, C., Bergantino, E., Lanfranchi, G., Valle, G., Carignani, G. and Frontali, L. (1992). A putative serine/threonine protein kinase gene on chromosome I11 of Saccharomyces cerevisiae. Yeast 8,71-77. Woolford, J. L. (1989). Nuclear pre-mRNA splicing in yeast. Yeast 5,439-457. Yannisch-Perron, C., Vieira, J. and Messing, J. (1989). Improved M 13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-1 19.
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The Complete Sequence of a 6146 bp Fragment of Saccharomyces cerevisiae Chromosome I11 Contains Two New Open-Reading Frames CATHAL WILSON, PAOLA GRISANTI AND LAURA FRONTALI* Department of Cellular and Developmental Biology, University of Rome 'La Sapienza', Piazzale Aldo Moro 5 , 00185 Rome, Italy
Received 30 January 1992; accepted 5 February 1992
As part of the EEC project to sequence the entire chromosome I11 of Saccharomyces cerevisiae we have sequenced a total of 1 1,040 bp from near the right end of the chromosome. A new protein kinase gene was found at one extremity of the sequenced region (Wilson et al., 1992), while the previously sequenced actin binding protein gene, ABPI, (Drubin et al., 1990) was found at the other extremity. We present here the sequence of the region between these two genes which has the potential to code for two new open reading frames (ORFs).
MATERIALS AND METHODS
DNA manipulations and sequencing strategy
Cloning, restriction analysis and preparation of plasmid DNA were essentially as described in Maniatis et al. (1982). A restriction map was constructed for the entire PM3712 insert and restriction fragments covering the left half of the insert were subcloned in both orientations in pTZl8R/pTZ19R. Directed overlapping deletions were generated using DNaseI (Lin et al., 1985) or exonuclease I11 (Henikoff, 1984). Sequencing of remaining gaps and across subclone Strains and plasmids boundaries was performed using oligonucleotideThe Escherichia coli strains used were JM101, directed sequencing of double-stranded DNA. Both JM109 and JM83 (Yannisch-Perron et al., 1989). double-stranded and single-stranded DNA were The S. cerevisiae strain W303-1B (MATa,ura,trp, used for sequencing using the chain-termination his,leu,ade) and its isogenic a and diploid derivatives method of Sanger et al. (1977). (R. Rothstein) were used for the gene disruption experiments. Subcloning, double-stranded DNA manipu- Gene disruption lations and production of single-stranded DNA The one-step gene-disruption technique was used were performed using the vectors pTZ18R/pTZ19R (Rothstein, 1983). A 489 bp SpeI fragment located (Pharmacia). The plasmid pFL39HIS' contains a between nucleotides 2062 and 2551 (Figure 1) was 1.76 kb insert carrying the yeast HIS3 gene and was replaced by the 1.76 kb fragment from pFL39HIS'. kindly provided by Professor P. Slonimski. Transformation of intact yeast cells was as described *Addressee for correspondence. by Ausubel et al. (1988) except that LiCl was used Chromosome 111DNA The lambda clone PM3712 (constructed by Professor M. Olson) contains an insert of 2&21 kb. Preparations of PM37 12 DNA were provided by Professor S. Oliver and Dr M. Gent. The insert DNA comes from the right end of chromosome I11 and is situated between the ABPI and rosl (cdc39) genes (Mortimer et al., 1989).
0749-503X/92/070569-07 $08.50 0 1992 by John Wiley & Sons Ltd
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ACMCMChTCACTGGMCTGCATTCMCTGMTCCACTATATCMTTACTA~GT~~CA~ACATATACATCCAGT~TACTMT 4548 T T T S V E V B S T E S T I S I T T V K P V T Y T S S D T N 1516
4549 1517
G G A W V \ C T G A T M C C A T M C A A ~ C C A C C ~ C T ~ M T T C C A T C A G T M C G A C ~ T M T M C G A G M C ~ G T f f i C C4638 ATM~~
4639 1547
G C A C C A M G C W V L C M C T A C G A C C I A T G T C G A ~ C G A ~ C C T C C A G T ~ A T T G C T A C T T ~ ~ ~ ~ G C A T C 4728 CT~CT
4729 1577
T G G A T T A ~ A C A C C C A T T G T C A G T A C G T A T G C T ~ C ~ G T ~ ~ C T ~ G T A G T M ~ ~ A T G A T M T 4818 ~MT~GATC 1606 U I T T P I V S T Y A G S A S K P L C S K P P U I U V U V I
4819 1607
M ~ C A ~ M ~ C C C G T A T A T A T T A T A T G T A C G T A T A T A T ~ A T A M C A ~ C A C T ~ A C T T A C C ~ M T M4908 T~~TGA N P I * * K G K I I S S S 1I5
G E L I T I T S S S Q T V I P S V T T I I T R T K V A I T S
A P K P T T T T Y V E Q R L S S S G I A T S P V A A A S S T
C C 4008 1336
4278 1426
1546
1576
4998 ~CA~A~CCCC~ 4909 ~ T C A G T C A ~ ~ A C T T C T T C C C C M ~ G T C A T C A T M T C A T A C C A ~ C A ~ A T C ~ ~ A ~ G G C A T P D T H T V E E G L N D D Y D Y W E N D E L Q C S U V K G S 145 174
T C M ~ ~ ~ T ~ ~ T C 4999 A C T T A G A G A M C T T C A A A C G ~ G T C ~ T ~ ~ ~ ~ M ~ C A C M C C T C T G C M 5088 S L S V E P T I T D P Y P K I L E C G R C D L S L P I P S D 115 144 CATCCTC 5089 T T ~ C A C C T T C G A T A M C C A T G ~ ~ C C ~ C A T ~ A T C T T ~ C C A ~ G T C G T C A C T C T G A T ~ ~ A M T A T T 5178 E K V K S L G H K K R V D K I K A W K D D S Q D T L Y E D E a5 114 5179 84
A A A A C C G C A C A C C T I M C T G A ~ T T ~ G M C ~ ~ A C A C T T C A T ~ ~ G A T ~ ~ A C ~ G A T G C C G G C A T5268 AGCATA 55 P A C L N V S L E K S C P K C K U L P S A T G K S A P U A Y
5269
T T C T T C A A A I G T A T T G A T G A T G A C T T T A G M T C G T G T M T T C C
54
E E P T N I I V K S D H L E R C S T C Q L D P A P E A R S E
5358 25
5359 24
G m G T A T T ~ C M T T G A A A C C f ~ T C A C ~ ~ G A T M T G 5448 1 T G N T N E I S V K T V R E S L T A K L V L P L P U YCR1104
5449
m ~ A T T ~ M ~ ~ C m ~ ~ C A T T A ~ ~ A T A M ~ ~ C T T G A C5538 G C T T ~ M ~ C A T
5539
T A C M G C M T G G A M C M M T T M G A l l M T G T A C G C M C W U G M C T A M
5629
CTAAATTATCTTATACTAGCMASCTT
5628
5655
Figure 1. Continued. Nucleotide and amino acid sequence ofYCRl102 and YCRl104. The stop codon TAA is represented by *. YCRl102 is coded for by the top strand while YCRl104 is coded for by the bottom strand.
SEQUENCE OF 6146 bp FRAGMENT OF S. CEREVISIAECHROMOSOMEI11
and boiled sonicated salmon sperm DNA was used as a carrier (Schiestl and Gietz, 1989). Total DNA was prepared from yeast cells as described by Ausubel et al. (1988).
573
similar to the conserved sequence elements GTAPyGT . . . . TACTAAC . . . . PyAG, which are necessary for the splicing of yeast introns (Woolford, 1989). However, the presence of a T instead of the invariable G (which is important for the correct splicing of the intron) at position 5 of the RESULTS AND DISCUSSION 5' element and the fact that such a splicing event Sequence analysis would disrupt the O R F makes it unlikely that it is an A comparison of our sequenced region with the intron. The YCRllO2 OR F could code for a large proGenEMBL database showed that the left-most part tein of 1609 amino acids. No significant homologies contained the actin binding protein gene A B P l were found with any proteins in the NBRF data(Drubin et al., 1990) beginning at amino acid base. The protein is rich in serine (26.3%) and number 15 of the ORF (data not shown). We found 12 differences between our sequence and the pub- threonine (18.1 %) residues. It also contains a large lished sequence (Table I), which are probably due to number of N-glycosylation sites (see below) which, strain polymorphism, as has been noted previously together with the possibility of 0-glycosylation on serine residues, may mean that the protein is in similar comparisons (e.g. Rad et al., 1991). heavily glycosylated. A hydrophobicity plot of the YCRl102 protein using the algorithm of Kyte and Table 1. Sequence changes in the ABPl gene Doolittle did not reveal any possible membranespanning regions. However, there is a hydrophobic N-terminal region and a very hydrophobic carboxy Nucleotide Previous New Amino acid tail (data not shown). position* sequence sequence change One of the most notable features of the YCRl102 ORF is the presence of a number of repeated 1674 C T S-rL regions, the most striking being an 81 amino acid 1990 T C almost-perfect repeat in the region between amino T 2436 A I+K acid residues 396 and 585 (repeat I . 1 and 1.2, Figure 3134 C T 2). Only three nucleotide changes (and one amino 3298 T C 3' acid change) occur in these repeats. Two other 3442 T C 3' repeated regions are also shown in Figure 2. Repeat 1921-1923 CCG TTC 3' 2.1 is in fact part of repeat 1 . 1 and 1.2. Thus this 19261928 TTC CAG 3' region is represented five times in the ORF. Figure 3 shows the locations of the repeated *Position numbers refer to the data of Drubin et nl. (1990). 3' regions, the 17 N-glycosylation sites and the 26 refers to the non-coding3' end of the sequence. cysteine residues in the YCRllO2 ORF. Of the 26 cysteine residues, 11 are accounted for by the The region between the A B P l gene and the repeated regions. Of the remaining 15 cysteines, ten KIN82 protein kinase gene (Wilson el al., 1992) are located in a short region of 77 amino acids. contains two ORFs, YCRl102 and YCRllO4, on These ten cysteines are arranged in five pairs with opposite strands, whose sequences are shown in four amino acid residues between the two cysteines Figure 1. The smaller of the ORFs, YCR1104, is of each pair. These cysteine residues might be preceded by a TATA box and a poly(dA-dT)-rich involved in intra- or intermolecular disulphide bond sequence which may act as a promoter element for formation and have a role in the stability of the constitutive transcription in yeast (Struhl, 1985). protein product of YCRl102. The presence of the The O RF could code for a protein of 182 amino majority of the N-glycosylation sites in the amino acids which has no remarkable features and which half of the protein and the majority of the cysteine did not reveal significant homology to any proteins residues in the carboxy half may represent two when compared with the NBRF database. different domains of the protein. One possibility is The upstream region of YCRl102 also contains that YCRllO2 codes for a cell wall protein. Cell wall a number of TATA boxes. Within the OR F the proteins are glycosylated, contain disulphide bonds sequence GTACTT . . . . TACTAAC . . . . CAG and some are rich in serine and threonine (Frevert (nucleotide positions 1880-2048 in Figure 1) is and Ballou, 1985).
574
C. WILSON ET AL.
Figure 2. Repeated regions in the YCRl102 open reading frame. Both the nucleotide and amino acid sequences areshownforrepeats 1.1 and 1.2.A blackdotsignifiesanucleotidechange. Repeat2.1 isapart ofrepeats 1.1 and 1.2.
REPEAT
-
6 6
Figure 3. Features of the YCR 1102 open reading frame. The repeated regions are shown by open boxes and the circled numbers. The locations of the cysteine residues are marked with a r and the N-glycosylation sites by A .
Gene disruption of YCR1102 The YCRllO2 gene was disrupted by replacing an internal 489 bp SpeI fragment with the HIS3 gene using the one-step gene-disruption method (Rothstein, 1983). Gene disruption was verified by Southern analysis (data not shown). No differences were found between the disrupted and nondisrupted cells when tested for growth rate,
mating, sporulation, spore viability, mitochondria1 stability, growth on different carbon sources, growth on high concentrations of NaC1, high and low temperature sensitivity and resistance to zymolyase or Kluyveromyces lactis killer toxin. Reduced stringency hybridization suggested that YCR1102 is present in a single copy in the S. cerevisiae genome (data not shown).
SEQUENCE OF 6146 bp FRAGMENT OF S. CEREMSIAE CHROMOSOME 111
ACKNOWLEDGEMENTS This work was supported by the C.N.R. and by Istituto Pasteur-Fondazione Cenci Bolognetti. C.W. was supported by Commission of the European Communities grant number BAP-0553-1. We thank the staff at MIPS for help with databank comparisons. REFERENCES Ausubel, F., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. (Eds) (1988). Current Protocols in Molecular Biology, vol. 2. John Wiley, New York. Drubin, D. G., Mulholland, J., Zhu, Z. and Botstein, D. (1990). Homology of a yeast actin-binding protein to signal transduction proteins and myosin-1. Nature 343, 288-290. Frevert, J. and Ballou, C. E. (1985). Saccharomyces cerevisiae structural cell wall mannoproteins. Biochemistry 24,753-759. Henikoff, S . (1984). Unidirectional digestion with exonuclease I11 creates targeted breakpoints for DNA sequencing. Gene 28,351-359. Lin, H., Lei, S. and Wilcox, G. (1985). An improved DNA sequencing strategy. Anal. Biochem. 147,114-1 19. Maniatis, T., Fritsch, E. F. and Sambrook, J. (Eds) (1982). Molecular Cloning. Cold Spring Harbor Laboratories, New York.
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Mortimer, R. K., Schild, D., Contopoulou, C. R. and Kans, J. A. (1989). Genetic and physical maps of Saccharomyces cerevisiae, edition 10. Yeast 5,321-403. Rad, M. R., Lutzenkirchen, K., Xu, G., Kleinhans, U. and Hollenberg, C. P. (1991). The complete sequence of a 11,953bp fragment from C l G on chromosome 111 encompasses four new open reading frames. Yeast 7, 533-538. Rothstein, R. (1983). Methods in Enzymology 101, 202-2 11. Sanger, F., Nicklen, S. and Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463-5467. Schiestl, R. H. and Gietz, R. D. (1989). High efficiency transformation of intact yeast cells using singlestranded nucleic acids as a carrier. Curr. Genet. 16, 339-346. Struhl, K. (1985). Naturally occurring poly(dA:dT) sequences are upstream promotor elements for constitutive transcription in yeast. Proc. Natl. Acad. Sci. USA 82,8419-8423. Wilson, C., Bergantino, E., Lanfranchi, G., Valle, G., Carignani, G. and Frontali, L. (1992). A putative serine/threonine protein kinase gene on chromosome I11 of Saccharomyces cerevisiae. Yeast 8,71-77. Woolford, J. L. (1989). Nuclear pre-mRNA splicing in yeast. Yeast 5,439-457. Yannisch-Perron, C., Vieira, J. and Messing, J. (1989). Improved M 13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-1 19.
