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Yeast Sequencing Reports
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Sequence of the sup61-RAD18 Region on Chromosome 111of Saccharomyces cerevisiae PAULE BENIT, ROLAND CHANET, FRANCIS FABRE, GERARD FAYE, HIROSHI FUKUHARA AND FREDERIC SOR* Institut Curie, Section de Biologie, Brit. 110, Centre Universitaire, 91405 Orsay, France
Received 7 October 1991; accepted 20 October 1991
A 7965 bp DNA segment from the right arm of chromosome I11 of Saccharomyces cerevisiae, encompassing the sup61 and RAD18 genes, was sequenced. Four new open reading frames were found in this DNA fragment. One of them, YCRlO3, is 51% homologous with the GI0 gene product of Xenopus laevis. KEY WORDS - Saccharomyces
cerevisiae; chromosome 111; sup6l; RAD18; chromosome sequencing; Zn finger
proteins.
INTRODUCTION In the framework of the European project for sequencing chromosome I11 of Saccharomyces cerevisiae, Vje have determined the sequence of a 7965 bp DNA segment encompassing the sup61 and RAD18 genes. This segment was subcloned from Lambda 5307 (Olson et al., 1986) prepared by S. Oliver (UMIST, Manchester). MATERIALS AND METHODS Restriction analysis and cloning in Escherichia coli were carried out as described in Maniatis et al. (1 982). All yeast genetic procedures and media were as described by Sherman et al. (1983). Gene replacements were done by the one-step disruption method described by Rothstein (1985). The yeast strains used were: MGD353-13D: atrpl ura3 arg4 leu2ade2 (A. Nicolas); MGD407: MATaIMATa trplltrpl ura3lura3 arg4/+ leu2/leu2 ade2/ /his3 /cyh2 (A. Nicolas); DG146-1: MATaIMATa ura3/ura3 leu2/leu2 trpl/trpl lys2/+ /his3 (this work). The major traits of the sequencing strategy used are as follows (Figure 1). The three EcoRI fragments of the region studied have been subcloned in
++
+
*To whom correspondence should be addressed. 0749-503) ~ f l F I G T G L C G L L T E Y K L N H U f l F E H f l f l K f l P O T D U U f l f l T P G V S flTGTTCflTflGGflflCCGGflTTGTGTGGGCTflCTCflCGGflGTflCflflGCTCflflCCRTTGGCGflTTCGflGCflTGCCCGCflflflCGGCCflCflGflCCGflTGTflGTTGCTGCCflCflCCGGGGTflCTCT 240 P N P F P f l F T l F U T G l L f l S O H f l O S S O F S T T l H T O U G V L L S V G CCflflflCCCGTTCCCCGCTTTCflCCflTflTTTTGGflCTGGGflTTCTGflTGTCCCRGCRCGCflCflGTCCTCGCflRTTTTCTflCTRCCflTTCflCRCGCflflTGGGGflTflCTTGTTGTCCTflTGGG
360
S F F R L L T F L l L F L U P N T N S f l f l S K P F T E L l T S F C L L C G G L U TCCTTCTTCCGTCTGCTflflCflTTTTTGflTTCTGTTTTTGGTGCCCflflCflCCflflCflGTGCCGCflTCCflflGCCTTTCflCGGflGTTGflTCflCCTCGTTCTGTCTCCTCTGTGGTGGTCTGGTfl
480
F ~ E S T D O S I E R ~ E ~ R G F T P ~ F T F N L ~ ~ G TTTflTGGflGTCCflCGGflTCflGTCCflTTGflflGCCflTGGflflTRTflGGGGGTTTflCCCCCflTGTTCflCTTTCflflCCTCflGTGTTGGflTTCGTTTCTTTGTTGflTGGCTTGGGflflflTGflTTTTfl
F ~ 600
~
L
L
~
F I U K O U L I K T R K T S L . TTTflTTTGGflflAGflCTGGCTCflTCfl~flflCCflGGflflflRCCflGTCTTTflRCCGCCflTflflTGGCTTTTCTTTTTTCTTTTTTTTTTCflCCCCCCCCTCflTCTTTGRGTGflGflflGCTTRTflTGC 720 TTTflCTTTTCCTCflCCTflCTTTTTflflTTCTTflflTTflTTflGTTTflTCCflGCflTTCflGTCflflTTCTGflflTGCGTTflTCGCTTCCTCflflRflTTTflTflflflflflTGflflTflflGCflflGflTCTCflTTGG 810 flflTTGflGTTCflflTGflCGflTTflTTGflGGGCCC~TflCCTGflTflflflflGTGflRTflTflTTflTGTRTflflTGflCTCTflCTGGflGGTTCTCTCTGflGflflTGTflflflflTflflTCCflCflflflflGflCflTGflflflfl 960 TTflGTTflCTCTflCflTflflTGTflTflflTflflGTTCTTCCTTCCTTTTTTCTTTTTTTflGTTTTTCTflCTCflCCflTCCTflCCTflCGCCflflGCTGCTCTflCTGTCTTGGflTCTGTGflflflflflflCGCC 1080
GGC~CT~TGGCCG~GTGGTT~~GGCG~G~GRCTCG~~-------------------RTCTCTTGGGCTC TflGflflflCflTGTflTflflflflCCTflCCGCTTTflflflCCflTflflTTTCflflflTCGflRflTGGCflCTflTGGCCGRGTGGTTflflGGCGRGflGflCTCGflflTGGflflTflflflflflGTTCGGCTflTCTCTTGGGCTC 1200 tRNRaer TGCCCGCGCTGGTTCR~~)~)TTCCTGCTGGTGTCG TGCCCGCGCTGGTTCflflflTCCTGCTGGTGTCGTTTflRTTTTTTTTRflflTflflCflTCGTTGflTTflflflflCflflTCCTflTTCflCTGCflGTCRCflGflCTTflGCflCflTTTflCflflCCflGTflCflflGflTC 1320 flflTCflCflCGCGTTGTGGTflGGflflCTTTGflCGflTGflTRCTGCGTTGGCTCTTGflflCGCflTflGTflCflTTTGTCGTTCflCflflCGGflflflGTGflflflTflCTGflflflRflflflflflGflflflflflTTTTCTTflG
I440
~ P ~ ~ I K T R R S K P R P O G F E K GGflCTCflTCflTCflflTGTflflRCflCCTCTflCflGCCGTCflflGflCTflGCRRflGCTCflCCTCflCflflflflCGflCflTGCCGCGCRTflRflGflCCflGflflGflTCCflflGCCTGCflCCTGflCGGGTTCGflflflfl 1560 VCfl903 ----> l K P T L T O F E l O L f l D R O K D K S S K L f l f l K S N E O L U E l l l O L H H O flflTCflflGCCflflCCCTCflCflGflTTTCGflflflTCCflflCTCflGflGflTGCCCflflflflGGflCflflGTCGTCTflflGCTCGCflGCflflflGTCCflflTGflGCflGCTCTGGGflGflTflflTGCflflCTCCflCCflCCfl 1680 f l S f l V l V T L V V K R K f l l S K D L V D U L l K E K $ ' f l D K L L l f l K U f l K T GCGC~TflCflTflTflTflCTCTGTflCTflCflflGflGflRflGGCCflTCTCCflflflGflCCTTTflCGflTTGGTTGflTflflflGGflflflflGTflTGCTGflTflflflTTGCTflflTTGCCflflflTGGCGCflflflflC Xba I G Y E K L C C L f l C l O K N E T N N G S T C I C f l U P R f l O L E E E f l f l K K G T CGGGTflTGflflflflflCTGTGCTGTCTGCGCTGCflTTCflflflflGflflCGflflflCTflflCflflCGGTflGCflCTTGCflTCTGCflGGGTGCCTCGTGCflCflGTTflGflGGflflGflflGCflCGCflflflflflGGGCflC
I800
1920
O U S F H O C U H C G C f l G C f l S T D . flCflGGTGTCCTTCCflTCRGTGCGTCCflCTGCCGTTGCflCGTTGCflGTflTTGlTCTGGCCflTGTflflTTflTCTCTCTCCCflflCGTTT
2010
TflGflflGflGGTTTTCGTCTCCTTCGTCflflflGflCGflCCflCTTTCCCflTTTTGGTCflTTGGflRCGCGflflRflflflCflflflflCflGflCGCGRGflRCGCGflCGCGflflflflflflTflTflTTTTTCCGTTTTTT TTTTTTTTCGGflflCflflflGRTRflCGCGTTRRflRCCTGTGGflTGTflflTflRGGflTCTGflflflCflCTGRCTRflflflCGCGTCCTCRTTTflflflflTCRflflflflflTTflCTTCTTCTCCTCCTTflCGGTGC CCTTTGRTCCTTCTCflflflCTTTflflflTflTflTGTTGCflTGflTTflflTTGflflflflTCflTRTflGflGTflTflTCTTflflCflGflGGTTCCflflCGCTTTflflflTCGTflTTCTflTTCTTTTCCCCCCTTTTflT
2160 2280 2100
f l R N E O l S l TflTflGTTflCflTCTflCTflTTTGflGCTTCTTTTflTTGflCCGflflflCTflCCTCTTflTTTCGflflGCflflflflflflflflflflflRflflTCRflCflflflflflTTCCflflCTTTTCflflTGflTGflflTGflflGflCflTflTCCfl 2520 YCR902 ----> I D G H N S F L T E K S T U L L T O f l K R T L E D E K E R l T P P S S T U f l K l TCflTTGflTGGCCflTflflTflGTTTTTTflflCGGflflflflflflGCflCCGTGCTflTTflflCCCflflGCCflflGflGflflCflCTflGRflGflCGflRflRGGflflflTGflTTflCTCC~CTCflflCTGTGflGflflflflfl 2610 flual R K E U N K f l P S H P L S P O H S S P l f l P S K f l K f l O f l S D T C f l f l S N G N L CflflTGflflGGflflGTflflflTRflGflGGCCGTCGCflTCCCCTCTCflCCGGflTCflCTCGTCCCCflflTTGCTCCflTCTflflGGCCflRGCGCCflflflGflTCGGflCflCflTGCGCTCGGTCCflflTGGTflflCC 2760 T L E E I L O S L E R ~ R I N G E L ~ K K P P ~ ~ ~ TflflCCTTGGflflGflflflTTCTTCflflTCTTTGGflflflGflflGflflGflflTflflflTGGTGflflCTCGCCflflGflflflCCTCCflTflTTCGTflTGCflflCTTTGflTTTGCTTGGCCflTTTTGCflflTCTCflGGflGG
~
K L T L S O l Y H U l H U H F P V V K O K D f l S U O N S I f l H N L S L N D f l F l GflflflGCTflflCGCTflTCCCflGflTflTflTCflTT~CGTTCflCTTCCCTTflTTflCflflGCflGflflflGflTGCTflGTTGGCflflRflTTCflflTflflGflCflTflflCTTGTCTTTflflflTGflTGCGTTCfl B a dI K T E K S C O G K G H F U E U R P G f l E T K F F K G E N f l G V E F U K D S L O D TCflflGflCTGflflRflGTCCTGCGflTGGTRflGGGTCflTTTCTGGGflGGTCflGflCCGGGTGCCGflflflCflflflflTTTTTCflflflGGTGflflRflTCGTGGTTflTGflflTTTGTflflflGGflCTCCTTflCflflG
~
L 2880
3000
3120
I G K V F E I O S T L D E L E O U E S G E G N D D L P D E E E R E E f l G K F P S flCflTTGGGflflGTflTTTTGflflflTflGflTTCTflCflCTTGflTGflflTTflGflflCflflGTTGflGflGTGGflGflflGGCflflTGflTGflTCTTCCTGflCGflGGflflGflflflGflGflGGflflGCflGGGflflflTTCCCTT
3210
l E l O L N S S P l L f l U S O L H H l P O L K T D N S U L N P H E N L E S f l f l N CCflTTGflflflTTCflflTTGflflCTCCTCCCCTflTflCTGflGflGTTTCCCflGTTflCflTCflCflTflCCGCflflTTGflflflflCflGflCflflCflGTGTflCTGRRCCCTCflCGflflflflCCTRG~GCGGfl
3360
I
~
L
~
I
150
P. BENIT ETAL.
U K N N N l T O f l N f l F N T L P l T S f l K S P O N F f l K V F T S F N S N F E O L GTGTGflflflflflCflflTflflTflTCflCCCflGGCflflRTflGflTTTflflTflCflCTCCCTflTflflCTflGCGCflflRGTCTCCTCflGflflTTTCflGflflflflTflTTTCflCCTCflTTCflflTTCflflflTTTTGflflGflTT 3600 S P L f l S N U G f l G S L L O P L P Y S P L K L Y O Q K N L f l L l l S K P O S O O S TflTCTCCflCTTCGflflGTflRTGTflGGGGCTGGTTCTCTflCTCGRCCCRCTTCCGTflTTCCCCflTTGRflGCTGTflCGflTCflGflflfl~flTCTTGCGCTCflTGTCGflflflCCflCflflTCTCflGCflflT
3720
Y S N S O L P P P P S S H G S D L L K T P K l l f l H S D G L E K T P S f l L l S T P CflTflTTCCflflTTCTCflflCTTCCflCCTCCflCCTTCCTCTCflTGGTTCGGflCTTflCTTflflflflCflCCCflflGflTGRGGCflTTCCGflTGGCTTRGflGflflflflCCCCflTCGCGGTTGflTflflGCflCflC
3810
K D G N S l L f l K U O T P S H L F E O L Y C S P L F f l f l I E T P I R ~ I T T P G 3960
CTflflGGflCGGTflflCTCGRTTTTGflGGflflflTGGCflGflCTCCTTCflCflCCTTTTTGflflGflTTTGTflCTGTTCTCCGCTflTTTflGflGCTflTflGflGflCTCCflflTCflGGTflTflTCflCGflCGCCGG
G N F G N P N F T K K U L C T f l C P H K f l N E F O I C F K f l f l U U f l G C L F C L GGGGCflflCTTTGGflflflCCCflflflTTTCflCCflflGflflflGTCCTCTGCflCCCGflTGTCCTCflCRRGCGCflRC~CflflflTTTGCTTCflflGCGGGCTGTTTGGCGTGGflTGTTTRTTCTGTT EcoR I E A R N . TGGflflGCGCGCflflCTGflflflflGflTTTCTGflTGGTflflCflflTflCTflCGGflTflGCflflTCflflflflRCflTCflTCCTTflTCflTflflTCflCCCTTCCflflCGflTflGCGGTflflTGflflflflGflflTTGflCTTTTT
1080
1200
TTTTTTTTTTTTTTTCflTTTTCTTTTCCTCflCCGflflRflCT~flTTflTTCTTflflTCTGflTGGflTTCflTCGCflflflCGGTTGGflTGRGTCCTflflflCTGGGCTGGflGTTflTCflTflTCflCflTflGCfl TTTTCTTTTTflATCTTflTTGTflTTGTCGGGTCflflCTRflCTTCTTTTCCCGCTCTTCTflTGGTflTRRTCRRTTGCflGflRGflGGCflGGflRflflRRTTGTflflTCflTflflCflflflflflTGflTflCflfl~T TTTTTTTCTflRGTTTTflTflTTTTflCflTflTCTTTflTTTTTTGflTCCTTflCGGflTTTTTflflTflCflGTflTTTflGCTGflflGTTTCCflTTCflCTTCflCTTflCCTTTTTGflTCTCTTTTflTTTTTfl CTTGCCCCGTTGCCTTGCflTTTTTCflflTflGflfl~GTGflflflflflCTflflGTflCCflCTTGflGCflflTGCCflCflTTflGflflGRflflRflCCflTCCGCflflGTGflGCflTCflCflGCTflCTflflGflflflflGGCCflT
1320 1110 1560 1680
N D H O l T T f l S D F T T T S l P S L Y O L D T L L f l C H l C K O F L K U 1800 TTTTflCTflCTCflTGGflCCflCCflflflTflflCCflCTGCflflGCGflCTTCflCGflCTflCTTCflRTflCCGflGCCTGTflCCflflTTGGflTflCflCTTTTGflGflTGTCflCflTTTGTflflflGflTTTTCTflflflflG RRDlO ----> P U L T P C G H T F C S L C l f l T H L N N O P N C P L C L F E F f l E S L L f l S E TCCCCGTCTTflflCRCCTTGTGGCCflTRCflTTTTGTTCCCTTTGTRTTRGRRCRC~TTTGflRTRRCCRRCCflRflTTGTCCTCTCTGCCTTTTCGRGTTCflGflGflGTCCTTGCTGflGflflGTG 4920 F L U S E l I O S Y T S L f l S S L L D R L f l I P K P T P U P E N E E U P G P E N flGTTCCTGGTCflGTGflflATflflTTCflflflGTTflTflCRTCCCTflCGflTCTTCCTT~CTflGflTGCflCTflflGGRTflCCGflflGCCTflCCCCTGTCCCTGflGflflTGflGGflflGTflCCflGGTCCTGflflfl 5010 S S U l E L l S E S E S O S U N f l f l O O D L O l U f l T S E f l K L f l K f l S l l T O l flTTCTTCRTGGflTflGflflCTCflTflTCflGflGTCTGflRflGTGflCflGTGTflRflTGCCGCTGflTGRTGflCTTGCflflRTTGTTGCflflCflflGTGflflflGflflflflCTTGCCflflflflGflTCCflTGflCTGflTfl
5160
L P L S S K P S K f l N F f l l l F f l S E f l I K K K S K P N E O l l f l O C P l C O O F V TflTTflCCflCTGflGTTCCflflflCCflTCCRflflflGGRflTTTTGCflflTGTTCflGflflGTGflflCGTflTCflflGflflflflflflTCflflRGCCflflflTGflflCflflflTGGCCCflGTGCCCCflTflTGTCflflCflflTTTT
5280
P L K f l L E K T H L O E C L T L O S L G K K P K l S T T F P T E S N P H N K S flTCCTCTTflflflGCCCTTGflflflflflflCflCflTTTGGflTG~flTGCCTRflCTTTflCflflTCflCTflGGCflflflflflflCCRflflflflTTTCTflCCflCTTTCCCTflCflGflGTCflflflTCCflCflTflflCflflflflGTT
S 5400
S R F K U R T P E U O K S S C G E T S H U O K Y L N S ~ ~ ~ ~ E H O ~ CflTCCflGflTTCflflGGTflCGA~CTCCflGflflGTCG~CflflflflGCTCflTGTGGTGRGflCCTCflCflTGTGGRTflflGTflTTTflflflCTCflflTGflTGflGTGCflGflRCflCCflflflGflTTGCCGflflGflTCfl
L P 5520
K
I
N
F T S N T O S O l K O K L S S L G L S T N G T R O N N I K f l Y N H Y E N L U N S 5610 flTTTTflCGTCTflTGflCTCflflTCCCflflATflflflflCflflflflflCTGTCflTCGTTGGGflCTGTCflflCTflflTGGTflCTflGGCflflflflCflTGflTTflflflflGflTflCflflTCflCTflCGflflflTGCTTTGEBBLL Ecofll N F C O S L E P U O E f l E L K f l O L L S U D U S H N K T P O N S S N K G G I S K 5760 ~TflflTTTTTGTGflTTCTCTflGflflCCTGTTGflTGflflGCTGflflCTflflflflflGflCflGTTGTTflflGCTGGGflTGTTTCflCflCflflTflflflflCCCCCCflflflflTflGTflGCflflCflflGGGTGGflflTTTCTfl L ~ ~ ~ K S N G K S S S V R K L L E N F K N O K F N R K G U ~ U ~ flflTTflflTGflTflflTGflflGflGTflflTGGGflflflTCTTCTTCflTflTflGGflflflTTflCTTGflflflflTTTCflflflflflCGflTflflflTTTflflTflGGflflflGGflTGGflTGGTTflTGTTTCGGflflGGflTTTTGCTfl
F
R K 5880
L I R E ~ K ~ K I K T G S S O S S G S U G H ~ N ~ G ~ G 6000 GGCTTflTCflGGGflflGCflflflflflTGflflflflTflflflflflCflGGTTCflTCGGflCflGTTCflGGTTCflGTGGGflCflTTCTflflTGflTGGflGflTGGTGTTGflRflflRGTTCflflflGTGflCCflGGGflflCCGflGG 0 0 l l E K O O D T U I N E D f l U f l G E f l N L P N E D S T O f l O L S f l E L l l O L N flTCflGCRflflTGGflGflflGGflTCflGGflCflCTGTTflTCflflCGflflGflTflGflGTTGCTGGTGflflflGflflflTTTGCCTflflCGflflGflTTCflflCTGflTGCTGflCTTflTCflflGflGflflTTflflTGGflCTTGfl
6120
E Y S K O P P G N N . flTGflflTflTflGTflAflGflCCCflCCCGGTflflCflflTTflflGGTGTGTflTflTGTflflTCflGGCCTGTTTGTTflGCTTGTGCRCflTTTGTTflflTTflflTflflTTTflTTTflflflflflTGGGCflflflflTflTflflflT
6210
GCflTflflflflGflTGflCflTflGRGflTflTGTGCflCflflflGCTflTGTflflTCflTflTTTflflCflTTflflTflTflTflTTflTTTCGTTflTTCflCflflTTCGTCflTGflflGGCCflGCGTflGTTTflCGGTTCCTGCTfl . L E O H L G f l Y N U T G f l
6360
CTGCATTflTCflTCGTTGflCflflTGTTTGTflTTCGflflTTflTTflTTCflflflGGTGCTGCflflTTGTTTGCGGflGGGGCCflCTGGflTTflGGCflflflGflflGTGflTCTCCflCGTTflflGTGCTGflflflTflfl U f l N O O N U l N T N S N N N L P f l f l l T O P P f l U P N P L S T l E U N L f l S l
6180
CTTCflCTflGflTflGflGflGflCGTCTTCGGTflflCflTCTflCCGTflCCRGflTGCCTGCflTTTCCGTCGTflflGCflTTCGGGflflflCflGGflGTflTTTflCflTTTTCGCTGflTTTTGTGGCGTflCCCCflfl U E S S L S U O E T U D U T G S f l O l l E T T L l l f l S U P T N U N E S l K H f l U G
6600
RCGCTTCflGflTGflflTCTTTTGflTGflCTGTflCTTCTflTCflTTflTTTCTTTCTGTflCflflflTCCTGflTGCflTCflflTTGflflCTflGCflGflTflTTTTGTGflGGGCTflCTTflCflflflflGflTGGTGCTG F f l E S S O K S S O U E l l l l E K O U F G S f l O I S S f l S I K H P S S U F S P f l
6720
TTGTGGTTRCflCCTGGCATCGGCTGflGCTflCTGTGCTTGflCGCGCTCTCCRCGGRTflRRGTTCCCTTGCCTflTflTCflGflCflGflGCGCCflflTTGflflGCflCCflCTflCTflCTflflflGflTCflTflG T T T U G P ~ P O R U T S S R S E U S L T G K G I O S L R G I S ~ G S S
S
6840 F I
O
~
~
F
E
~
K
R
~
151
SEQUENCE OF supbl-RAD 18 REGION ON CHROMOSOME 111 flTGCCflTflGflflGTCCCTTCflflTflTTTGTGGflTTCTGflTflGGGflflGflTTGflCCflRCflCTCGTCTTflGGTGflTTGTflCflGCflTCflGTGflRflflCflCTflGTGGGTTCCGflflCTGTCflGCCflflflR S f l M S T G E l N T S E S L S S O G U S T K P S O U f l O T F U S T P E S S D f l L
6960
CflCTflGTGGGTTCCGflflTTGTCflGCCflRflflCflCTflGTGGGTTCCflflflTTGTCflGCCflflCflCflCTGGTGTflTTCCGflflTTflTCflflTCflflGGCflCTGGflGCGTTCTGflflTCCTCGGTflGflGfl 7080 U S T P E S ~ D f l L U S T P E L N D f l L U S T Y E S N D l L f l S S f l E S D E T S flflGCflflTTflCTCTCGflflGTCTTCGTTTClGTTTTTGflGflflTGflGCTflGTTGCCGTCTCCflCflflTflGCTGflTGflflflCflGGTTCflGflGflTGGflflTGflflflTTTflGflflflflflGTGGflflTCTGflGfl F f l I U R S T K T E T K S F S S T f l T E U I f l S S U P E S I S H F K S F T S D
7200 S
TTGflflGGGflTTGflTTCflGflflGCflflGflflCflGflflGflflGTTflCflGTCGflTflCTTCTflTTGflCTGGflflGCCflGflGGTTflTflGflTGflflTCflGflflflflTGTflCTCGGflTGCGflGGflflflTflGflGTCCT l S P l S E S f l L U S S T U T S U E l S O F G S T I S S D S F T S P H S S l S 0
7320
GGGflTGCTGflTGTTGflCflflflflTTTCTTCflTTTGGGflTCflflflTTflGflCGCflflCTGflGTCflflflTTCGGflTGCTflTflGflflflTflGflflTGCGTflGGTGTTTCflflCCflTflCGCTCTflflTTCTGflTG Q S f l S T S L l E E N P l L N S f l U S D F E S f l I S I S H T P T E U ~ f l E L
E
CTflTGGGflTCflflTTTCGGflGflCGGGCGflflGflflflCCflTflGTGTCTGTGGGCGTCflflCflflCflTTGflflGflflGflTGTTGTflflCflTflCTGCTCGGCTTCTGCflTTGGflflTCTGflTGCflGGTflTflT ~ I P D I E S U P S S U ~ T D T P T L L ~
~
7440
S 7560 ~
TCCTflTflCTCflflTTTTTGTflflTGflCTTCflGTGflTTflTTTTTGTCTTflTflflflTCTCTCTCGTGGflTGCCGGTTTTGflGTTGTCTflGflRflflGflTGTGTTGGCTGCRTCflTTGGTGGGGTTTG N f l Y E l K T l U E T l I K T K Y I E f l T S f l P K S N O L F S T N f l f l D N T P N
7680
TTTGTGTGGTGGTflflCCGCflGTCGCGGTflCTTGTGGflTflflTGCTGTflGTflGGGCTflGTTGTTflflCGflTGflGGflflflGflGflTflflTGflflGflTGflflGflTGflflGflCGGCflTGTGTGflTflGCGflflG T ~ T T T ~ ~ T ~ T S T S L ~ T T P S T T
7800 ~
L
GTGflTTCflGTflCTTflflGflGTTTTGflflflCGCTTTCTCTflTCTGTTGTTflTTTCGCTTTCTGflTTGTTCTflCTflTTflflCGflGGflCflflTTTCTCCTCCflflGTCflGGflGTCGGCTCflGGflTflTT P S E T S L L K S U S E f l D T T l E S E S Q E U l L S S L K E E L D P T P E P Y
7920
CTGTGflTflGflflTTTGflflGflTGflGGflTflflCflflflTCTflTCGflCTCTGaa::c E T I S N S S S S L L D I S E (---YCfl9Ol
Figure 3. Nucleotide sequence of the sequenced region. The deduced amino acid sequences of the RAD18 gene and the open reading frames are indicated above or below. The arrow next to the name of the reading frames indicates the direction of transcription. The sequence of the mature serine tRNA is indicated in italics. Some restriction sites relevant to our studies are underlined.
-
YCR904
1000
2000
3000
4000
+ YCR903
YCR902
-5000
RAD18
6000
7000
aooo
YCR901
Figure 4. Identification of open reading frames with the 7965 bp fragment using DNA Strider software (Marck, 1988). Long barsare stop codons and short bars are ATG codons.
homology with published sequences. YCR902 was disrupted by insertion of the LEU2 gene between the AvaI (nucleotide 2618) and BarnHI (nucleotide 291 1) sites. This disruption was introduced by gene replacement in haploid strain MGD353-13D. The disruption was tested for the following auxotrophies: tyr, his, ile, lys, met, phe, thr and for
the growth on glycerol-complete agar plate. No differences were observed between the disrupted and control strains. RAD18 (bases 4692 to 6152) The RADI8 gene of S. cerevisiae is involved in the error-prone DNA repair. It had previously been
~
~
~
~
~
~
L
~
152
P. BENIT ET AL.
60 903 MPRIXTRRSXPAPDGFEXIXPTLTDFEIQLRDAQWXSSKLAAXSNEQLWEIMQLHHQRS
.. .. .. .. .. .. .. .. .. .. .. .. .. ..
.. .. ..
.. .. .. ..
... ...
G10 MPKVK-RSRKPPPDGWELIEPTLDELDQ~TD--PHEGKRKVESLWPIFRIHHQXT
102 903 RYIYTLYYKRKAISKDLYDWLIKEXYADXLLIAKWRKTGYEKLCCLRCIQXNETNNGSTC
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ....................................
.. .. .. ..
G10 RYIFDLFYXRXAISRELMYCIREGYADXNLIAKWKKQGYENLCCLRCIQTRDTNFGTNC 150 903 ~RVPRAQLEEEARKXGTQVSFHQCVHCGCRGCASTD
..... .....
.. ..
.. .. .. .. .. .. .. ..
G10 ICRVPXTXLEVG--------RIIECTHCGCRGCSG Figure 5.Comparison of protein YCR903 with protein GI0 of Xenopus (McGrew et al., 1989). The zincfinger domain is underlined.
sequenced by two different laboratories, including ours (Chanet et al., 1988; Jones et al., 1988; Fabre et al., 1989). The only difference found in this new determination was an additional T residue in a long stretch of T (position 4200) located between the 3’ end of YCR902 and the 5‘ end of RAD18. YCR90I (bases 6320 to 7965) This sequence is the 3’ end of an O R F that is 1065 amino acids long. The 5‘ end of this gene was sequenced by Fiers’s group (University of Gent). This protein is rich in serine and presents a repeating motif of ten amino acid residues (ADNSEPTSVL), which is repeated six times. It also contains the HDEL amino acid sequence typical of proteins associated with the endoplasmic reticulum (G. Lauquin, personal communication). The deletion of the 3’ end of this gene (the two adjacent 1607 and 2329 bp long EcoRI fragments were deleted and replaced by the LEU2 gene; Fabre et al., 1989) was not lethal in haploid cells and expressed no mutant phenotype. As judged by Northern blot analysis, the YCR901 transcript is about 3.35 kb long (F. Fabre, unpublished result). ACKNOWLEDGEMENTS This work was supported in part by the Commission of the European Communities (Biotechnology Action Program). REFERENCES Brandriss, M. C., Soll, L. and Botstein, D. (1975). Recessive lethal amber suppressors in yeast. Genetics 79,551-560.
Brandriss, M. C., Steward, J. W., Sherman, F. and Botstein, D. (1976). Substitution of serine caused by a recessive lethal suppressor in yeast. J . Mol. Biol. 102, 467476. Chanet, R., Magana-Schwencke, N. and Fabre, F. (1988). Potential DNA-binding domains in the RAD18 gene product of Saccharomyces cerevisiae. Gene 74, 543-547. Fabre, F., Magana-Schwencke, N. and Chanet, R. (1989). Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of radl8 deletion mutants. Mol. Gen. Genet. 215,425430. Jones, J. S., Weber, S . and Prakash, L. (1988). The Saccharomyces cerevisiae RAD18 gene encodes a protein that contains potential zinc finger domains for nucleic acid binding and a putative nucleotide binding sequence. Nucl. Acids Res. 16,7 1 19-7 13 1. Lin, H. C., Lei, S. P. and Wilcox, G. (1983). An improved DNA sequencing strategy. Anal. Biochem. 147, 114-1 19. Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982). Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Marck, C. (1988). DNA strider: a C program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucl. Acids Res. 16, 1829-1836. McGrew, L., Dworkin-Rastl, E., Dworkin, M. and Richter, J. (1989). Poly(A) elongation during Xenopus oocyte maturation is required for translational recruitment and is mediated by a short sequence element. Genes Dev. 3,803-8 15. Mortimer, R. K. and Hawthorne, D. C. (1973). Genetic mapping in Saccharomyces IV. Mapping of temperature-sensitive genes and use of disomic strains in localizing genes. Genetics 74,33-54. Mortimer, R. K., Schild, D., Contopoulou, C. R. and Kaus, J. A. (1989). Genetic map of Saccharomyces cerevisiae. Edition 10. Yeast 5,321403.
SEQUENCE OF sup61-RAD 18 REGION ON CHROMOSOME I11
Olson, M. V., Page, G. S., Sentenac, A., Piper, P. W., Worthington, M., Weiss, R. B. and Hall, B. D. (1981). Only one of two closely related yeast suppressor tRNA genes contains an intervening sequence. Nature 291, 464469.
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
153 strategy for genomic restriction mapping in yeast. Proc. Natl. Acad. Sci. USA 74,5463-5467. Rothstein, R. (1985). Cloning in yeast. In Glover, D. M. (Ed.), DNA Cloning. Volume II, A Practical Approach. IRL Press, New York, pp. 45-66. Sherman, F., Fink, G . and Hicks, J. (1983). Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
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Sequence of the sup61-RAD18 Region on Chromosome 111of Saccharomyces cerevisiae PAULE BENIT, ROLAND CHANET, FRANCIS FABRE, GERARD FAYE, HIROSHI FUKUHARA AND FREDERIC SOR* Institut Curie, Section de Biologie, Brit. 110, Centre Universitaire, 91405 Orsay, France
Received 7 October 1991; accepted 20 October 1991
A 7965 bp DNA segment from the right arm of chromosome I11 of Saccharomyces cerevisiae, encompassing the sup61 and RAD18 genes, was sequenced. Four new open reading frames were found in this DNA fragment. One of them, YCRlO3, is 51% homologous with the GI0 gene product of Xenopus laevis. KEY WORDS - Saccharomyces
cerevisiae; chromosome 111; sup6l; RAD18; chromosome sequencing; Zn finger
proteins.
INTRODUCTION In the framework of the European project for sequencing chromosome I11 of Saccharomyces cerevisiae, Vje have determined the sequence of a 7965 bp DNA segment encompassing the sup61 and RAD18 genes. This segment was subcloned from Lambda 5307 (Olson et al., 1986) prepared by S. Oliver (UMIST, Manchester). MATERIALS AND METHODS Restriction analysis and cloning in Escherichia coli were carried out as described in Maniatis et al. (1 982). All yeast genetic procedures and media were as described by Sherman et al. (1983). Gene replacements were done by the one-step disruption method described by Rothstein (1985). The yeast strains used were: MGD353-13D: atrpl ura3 arg4 leu2ade2 (A. Nicolas); MGD407: MATaIMATa trplltrpl ura3lura3 arg4/+ leu2/leu2 ade2/ /his3 /cyh2 (A. Nicolas); DG146-1: MATaIMATa ura3/ura3 leu2/leu2 trpl/trpl lys2/+ /his3 (this work). The major traits of the sequencing strategy used are as follows (Figure 1). The three EcoRI fragments of the region studied have been subcloned in
++
+
*To whom correspondence should be addressed. 0749-503) ~ f l F I G T G L C G L L T E Y K L N H U f l F E H f l f l K f l P O T D U U f l f l T P G V S flTGTTCflTflGGflflCCGGflTTGTGTGGGCTflCTCflCGGflGTflCflflGCTCflflCCRTTGGCGflTTCGflGCflTGCCCGCflflflCGGCCflCflGflCCGflTGTflGTTGCTGCCflCflCCGGGGTflCTCT 240 P N P F P f l F T l F U T G l L f l S O H f l O S S O F S T T l H T O U G V L L S V G CCflflflCCCGTTCCCCGCTTTCflCCflTflTTTTGGflCTGGGflTTCTGflTGTCCCRGCRCGCflCflGTCCTCGCflRTTTTCTflCTRCCflTTCflCRCGCflflTGGGGflTflCTTGTTGTCCTflTGGG
360
S F F R L L T F L l L F L U P N T N S f l f l S K P F T E L l T S F C L L C G G L U TCCTTCTTCCGTCTGCTflflCflTTTTTGflTTCTGTTTTTGGTGCCCflflCflCCflflCflGTGCCGCflTCCflflGCCTTTCflCGGflGTTGflTCflCCTCGTTCTGTCTCCTCTGTGGTGGTCTGGTfl
480
F ~ E S T D O S I E R ~ E ~ R G F T P ~ F T F N L ~ ~ G TTTflTGGflGTCCflCGGflTCflGTCCflTTGflflGCCflTGGflflTRTflGGGGGTTTflCCCCCflTGTTCflCTTTCflflCCTCflGTGTTGGflTTCGTTTCTTTGTTGflTGGCTTGGGflflflTGflTTTTfl
F ~ 600
~
L
L
~
F I U K O U L I K T R K T S L . TTTflTTTGGflflAGflCTGGCTCflTCfl~flflCCflGGflflflRCCflGTCTTTflRCCGCCflTflflTGGCTTTTCTTTTTTCTTTTTTTTTTCflCCCCCCCCTCflTCTTTGRGTGflGflflGCTTRTflTGC 720 TTTflCTTTTCCTCflCCTflCTTTTTflflTTCTTflflTTflTTflGTTTflTCCflGCflTTCflGTCflflTTCTGflflTGCGTTflTCGCTTCCTCflflRflTTTflTflflflflflTGflflTflflGCflflGflTCTCflTTGG 810 flflTTGflGTTCflflTGflCGflTTflTTGflGGGCCC~TflCCTGflTflflflflGTGflRTflTflTTflTGTRTflflTGflCTCTflCTGGflGGTTCTCTCTGflGflflTGTflflflflTflflTCCflCflflflflGflCflTGflflflfl 960 TTflGTTflCTCTflCflTflflTGTflTflflTflflGTTCTTCCTTCCTTTTTTCTTTTTTTflGTTTTTCTflCTCflCCflTCCTflCCTflCGCCflflGCTGCTCTflCTGTCTTGGflTCTGTGflflflflflflCGCC 1080
GGC~CT~TGGCCG~GTGGTT~~GGCG~G~GRCTCG~~-------------------RTCTCTTGGGCTC TflGflflflCflTGTflTflflflflCCTflCCGCTTTflflflCCflTflflTTTCflflflTCGflRflTGGCflCTflTGGCCGRGTGGTTflflGGCGRGflGflCTCGflflTGGflflTflflflflflGTTCGGCTflTCTCTTGGGCTC 1200 tRNRaer TGCCCGCGCTGGTTCR~~)~)TTCCTGCTGGTGTCG TGCCCGCGCTGGTTCflflflTCCTGCTGGTGTCGTTTflRTTTTTTTTRflflTflflCflTCGTTGflTTflflflflCflflTCCTflTTCflCTGCflGTCRCflGflCTTflGCflCflTTTflCflflCCflGTflCflflGflTC 1320 flflTCflCflCGCGTTGTGGTflGGflflCTTTGflCGflTGflTRCTGCGTTGGCTCTTGflflCGCflTflGTflCflTTTGTCGTTCflCflflCGGflflflGTGflflflTflCTGflflflRflflflflflGflflflflflTTTTCTTflG
I440
~ P ~ ~ I K T R R S K P R P O G F E K GGflCTCflTCflTCflflTGTflflRCflCCTCTflCflGCCGTCflflGflCTflGCRRflGCTCflCCTCflCflflflflCGflCflTGCCGCGCRTflRflGflCCflGflflGflTCCflflGCCTGCflCCTGflCGGGTTCGflflflfl 1560 VCfl903 ----> l K P T L T O F E l O L f l D R O K D K S S K L f l f l K S N E O L U E l l l O L H H O flflTCflflGCCflflCCCTCflCflGflTTTCGflflflTCCflflCTCflGflGflTGCCCflflflflGGflCflflGTCGTCTflflGCTCGCflGCflflflGTCCflflTGflGCflGCTCTGGGflGflTflflTGCflflCTCCflCCflCCfl 1680 f l S f l V l V T L V V K R K f l l S K D L V D U L l K E K $ ' f l D K L L l f l K U f l K T GCGC~TflCflTflTflTflCTCTGTflCTflCflflGflGflRflGGCCflTCTCCflflflGflCCTTTflCGflTTGGTTGflTflflflGGflflflflGTflTGCTGflTflflflTTGCTflflTTGCCflflflTGGCGCflflflflC Xba I G Y E K L C C L f l C l O K N E T N N G S T C I C f l U P R f l O L E E E f l f l K K G T CGGGTflTGflflflflflCTGTGCTGTCTGCGCTGCflTTCflflflflGflflCGflflflCTflflCflflCGGTflGCflCTTGCflTCTGCflGGGTGCCTCGTGCflCflGTTflGflGGflflGflflGCflCGCflflflflflGGGCflC
I800
1920
O U S F H O C U H C G C f l G C f l S T D . flCflGGTGTCCTTCCflTCRGTGCGTCCflCTGCCGTTGCflCGTTGCflGTflTTGlTCTGGCCflTGTflflTTflTCTCTCTCCCflflCGTTT
2010
TflGflflGflGGTTTTCGTCTCCTTCGTCflflflGflCGflCCflCTTTCCCflTTTTGGTCflTTGGflRCGCGflflRflflflCflflflflCflGflCGCGRGflRCGCGflCGCGflflflflflflTflTflTTTTTCCGTTTTTT TTTTTTTTCGGflflCflflflGRTRflCGCGTTRRflRCCTGTGGflTGTflflTflRGGflTCTGflflflCflCTGRCTRflflflCGCGTCCTCRTTTflflflflTCRflflflflflTTflCTTCTTCTCCTCCTTflCGGTGC CCTTTGRTCCTTCTCflflflCTTTflflflTflTflTGTTGCflTGflTTflflTTGflflflflTCflTRTflGflGTflTflTCTTflflCflGflGGTTCCflflCGCTTTflflflTCGTflTTCTflTTCTTTTCCCCCCTTTTflT
2160 2280 2100
f l R N E O l S l TflTflGTTflCflTCTflCTflTTTGflGCTTCTTTTflTTGflCCGflflflCTflCCTCTTflTTTCGflflGCflflflflflflflflflflflRflflTCRflCflflflflflTTCCflflCTTTTCflflTGflTGflflTGflflGflCflTflTCCfl 2520 YCR902 ----> I D G H N S F L T E K S T U L L T O f l K R T L E D E K E R l T P P S S T U f l K l TCflTTGflTGGCCflTflflTflGTTTTTTflflCGGflflflflflflGCflCCGTGCTflTTflflCCCflflGCCflflGflGflflCflCTflGRflGflCGflRflRGGflflflTGflTTflCTCC~CTCflflCTGTGflGflflflflfl 2610 flual R K E U N K f l P S H P L S P O H S S P l f l P S K f l K f l O f l S D T C f l f l S N G N L CflflTGflflGGflflGTflflflTRflGflGGCCGTCGCflTCCCCTCTCflCCGGflTCflCTCGTCCCCflflTTGCTCCflTCTflflGGCCflRGCGCCflflflGflTCGGflCflCflTGCGCTCGGTCCflflTGGTflflCC 2760 T L E E I L O S L E R ~ R I N G E L ~ K K P P ~ ~ ~ TflflCCTTGGflflGflflflTTCTTCflflTCTTTGGflflflGflflGflflGflflTflflflTGGTGflflCTCGCCflflGflflflCCTCCflTflTTCGTflTGCflflCTTTGflTTTGCTTGGCCflTTTTGCflflTCTCflGGflGG
~
K L T L S O l Y H U l H U H F P V V K O K D f l S U O N S I f l H N L S L N D f l F l GflflflGCTflflCGCTflTCCCflGflTflTflTCflTT~CGTTCflCTTCCCTTflTTflCflflGCflGflflflGflTGCTflGTTGGCflflRflTTCflflTflflGflCflTflflCTTGTCTTTflflflTGflTGCGTTCfl B a dI K T E K S C O G K G H F U E U R P G f l E T K F F K G E N f l G V E F U K D S L O D TCflflGflCTGflflRflGTCCTGCGflTGGTRflGGGTCflTTTCTGGGflGGTCflGflCCGGGTGCCGflflflCflflflflTTTTTCflflflGGTGflflRflTCGTGGTTflTGflflTTTGTflflflGGflCTCCTTflCflflG
~
L 2880
3000
3120
I G K V F E I O S T L D E L E O U E S G E G N D D L P D E E E R E E f l G K F P S flCflTTGGGflflGTflTTTTGflflflTflGflTTCTflCflCTTGflTGflflTTflGflflCflflGTTGflGflGTGGflGflflGGCflflTGflTGflTCTTCCTGflCGflGGflflGflflflGflGflGGflflGCflGGGflflflTTCCCTT
3210
l E l O L N S S P l L f l U S O L H H l P O L K T D N S U L N P H E N L E S f l f l N CCflTTGflflflTTCflflTTGflflCTCCTCCCCTflTflCTGflGflGTTTCCCflGTTflCflTCflCflTflCCGCflflTTGflflflflCflGflCflflCflGTGTflCTGRRCCCTCflCGflflflflCCTRG~GCGGfl
3360
I
~
L
~
I
150
P. BENIT ETAL.
U K N N N l T O f l N f l F N T L P l T S f l K S P O N F f l K V F T S F N S N F E O L GTGTGflflflflflCflflTflflTflTCflCCCflGGCflflRTflGflTTTflflTflCflCTCCCTflTflflCTflGCGCflflRGTCTCCTCflGflflTTTCflGflflflflTflTTTCflCCTCflTTCflflTTCflflflTTTTGflflGflTT 3600 S P L f l S N U G f l G S L L O P L P Y S P L K L Y O Q K N L f l L l l S K P O S O O S TflTCTCCflCTTCGflflGTflRTGTflGGGGCTGGTTCTCTflCTCGRCCCRCTTCCGTflTTCCCCflTTGRflGCTGTflCGflTCflGflflfl~flTCTTGCGCTCflTGTCGflflflCCflCflflTCTCflGCflflT
3720
Y S N S O L P P P P S S H G S D L L K T P K l l f l H S D G L E K T P S f l L l S T P CflTflTTCCflflTTCTCflflCTTCCflCCTCCflCCTTCCTCTCflTGGTTCGGflCTTflCTTflflflflCflCCCflflGflTGRGGCflTTCCGflTGGCTTRGflGflflflflCCCCflTCGCGGTTGflTflflGCflCflC
3810
K D G N S l L f l K U O T P S H L F E O L Y C S P L F f l f l I E T P I R ~ I T T P G 3960
CTflflGGflCGGTflflCTCGRTTTTGflGGflflflTGGCflGflCTCCTTCflCflCCTTTTTGflflGflTTTGTflCTGTTCTCCGCTflTTTflGflGCTflTflGflGflCTCCflflTCflGGTflTflTCflCGflCGCCGG
G N F G N P N F T K K U L C T f l C P H K f l N E F O I C F K f l f l U U f l G C L F C L GGGGCflflCTTTGGflflflCCCflflflTTTCflCCflflGflflflGTCCTCTGCflCCCGflTGTCCTCflCRRGCGCflRC~CflflflTTTGCTTCflflGCGGGCTGTTTGGCGTGGflTGTTTRTTCTGTT EcoR I E A R N . TGGflflGCGCGCflflCTGflflflflGflTTTCTGflTGGTflflCflflTflCTflCGGflTflGCflflTCflflflflRCflTCflTCCTTflTCflTflflTCflCCCTTCCflflCGflTflGCGGTflflTGflflflflGflflTTGflCTTTTT
1080
1200
TTTTTTTTTTTTTTTCflTTTTCTTTTCCTCflCCGflflRflCT~flTTflTTCTTflflTCTGflTGGflTTCflTCGCflflflCGGTTGGflTGRGTCCTflflflCTGGGCTGGflGTTflTCflTflTCflCflTflGCfl TTTTCTTTTTflATCTTflTTGTflTTGTCGGGTCflflCTRflCTTCTTTTCCCGCTCTTCTflTGGTflTRRTCRRTTGCflGflRGflGGCflGGflRflflRRTTGTflflTCflTflflCflflflflflTGflTflCflfl~T TTTTTTTCTflRGTTTTflTflTTTTflCflTflTCTTTflTTTTTTGflTCCTTflCGGflTTTTTflflTflCflGTflTTTflGCTGflflGTTTCCflTTCflCTTCflCTTflCCTTTTTGflTCTCTTTTflTTTTTfl CTTGCCCCGTTGCCTTGCflTTTTTCflflTflGflfl~GTGflflflflflCTflflGTflCCflCTTGflGCflflTGCCflCflTTflGflflGRflflRflCCflTCCGCflflGTGflGCflTCflCflGCTflCTflflGflflflflGGCCflT
1320 1110 1560 1680
N D H O l T T f l S D F T T T S l P S L Y O L D T L L f l C H l C K O F L K U 1800 TTTTflCTflCTCflTGGflCCflCCflflflTflflCCflCTGCflflGCGflCTTCflCGflCTflCTTCflRTflCCGflGCCTGTflCCflflTTGGflTflCflCTTTTGflGflTGTCflCflTTTGTflflflGflTTTTCTflflflflG RRDlO ----> P U L T P C G H T F C S L C l f l T H L N N O P N C P L C L F E F f l E S L L f l S E TCCCCGTCTTflflCRCCTTGTGGCCflTRCflTTTTGTTCCCTTTGTRTTRGRRCRC~TTTGflRTRRCCRRCCflRflTTGTCCTCTCTGCCTTTTCGRGTTCflGflGflGTCCTTGCTGflGflflGTG 4920 F L U S E l I O S Y T S L f l S S L L D R L f l I P K P T P U P E N E E U P G P E N flGTTCCTGGTCflGTGflflATflflTTCflflflGTTflTflCRTCCCTflCGflTCTTCCTT~CTflGflTGCflCTflflGGRTflCCGflflGCCTflCCCCTGTCCCTGflGflflTGflGGflflGTflCCflGGTCCTGflflfl 5010 S S U l E L l S E S E S O S U N f l f l O O D L O l U f l T S E f l K L f l K f l S l l T O l flTTCTTCRTGGflTflGflflCTCflTflTCflGflGTCTGflRflGTGflCflGTGTflRflTGCCGCTGflTGRTGflCTTGCflflRTTGTTGCflflCflflGTGflflflGflflflflCTTGCCflflflflGflTCCflTGflCTGflTfl
5160
L P L S S K P S K f l N F f l l l F f l S E f l I K K K S K P N E O l l f l O C P l C O O F V TflTTflCCflCTGflGTTCCflflflCCflTCCRflflflGGRflTTTTGCflflTGTTCflGflflGTGflflCGTflTCflflGflflflflflflTCflflRGCCflflflTGflflCflflflTGGCCCflGTGCCCCflTflTGTCflflCflflTTTT
5280
P L K f l L E K T H L O E C L T L O S L G K K P K l S T T F P T E S N P H N K S flTCCTCTTflflflGCCCTTGflflflflflflCflCflTTTGGflTG~flTGCCTRflCTTTflCflflTCflCTflGGCflflflflflflCCRflflflflTTTCTflCCflCTTTCCCTflCflGflGTCflflflTCCflCflTflflCflflflflGTT
S 5400
S R F K U R T P E U O K S S C G E T S H U O K Y L N S ~ ~ ~ ~ E H O ~ CflTCCflGflTTCflflGGTflCGA~CTCCflGflflGTCG~CflflflflGCTCflTGTGGTGRGflCCTCflCflTGTGGRTflflGTflTTTflflflCTCflflTGflTGflGTGCflGflRCflCCflflflGflTTGCCGflflGflTCfl
L P 5520
K
I
N
F T S N T O S O l K O K L S S L G L S T N G T R O N N I K f l Y N H Y E N L U N S 5610 flTTTTflCGTCTflTGflCTCflflTCCCflflATflflflflCflflflflflCTGTCflTCGTTGGGflCTGTCflflCTflflTGGTflCTflGGCflflflflCflTGflTTflflflflGflTflCflflTCflCTflCGflflflTGCTTTGEBBLL Ecofll N F C O S L E P U O E f l E L K f l O L L S U D U S H N K T P O N S S N K G G I S K 5760 ~TflflTTTTTGTGflTTCTCTflGflflCCTGTTGflTGflflGCTGflflCTflflflflflGflCflGTTGTTflflGCTGGGflTGTTTCflCflCflflTflflflflCCCCCCflflflflTflGTflGCflflCflflGGGTGGflflTTTCTfl L ~ ~ ~ K S N G K S S S V R K L L E N F K N O K F N R K G U ~ U ~ flflTTflflTGflTflflTGflflGflGTflflTGGGflflflTCTTCTTCflTflTflGGflflflTTflCTTGflflflflTTTCflflflflflCGflTflflflTTTflflTflGGflflflGGflTGGflTGGTTflTGTTTCGGflflGGflTTTTGCTfl
F
R K 5880
L I R E ~ K ~ K I K T G S S O S S G S U G H ~ N ~ G ~ G 6000 GGCTTflTCflGGGflflGCflflflflflTGflflflflTflflflflflCflGGTTCflTCGGflCflGTTCflGGTTCflGTGGGflCflTTCTflflTGflTGGflGflTGGTGTTGflRflflRGTTCflflflGTGflCCflGGGflflCCGflGG 0 0 l l E K O O D T U I N E D f l U f l G E f l N L P N E D S T O f l O L S f l E L l l O L N flTCflGCRflflTGGflGflflGGflTCflGGflCflCTGTTflTCflflCGflflGflTflGflGTTGCTGGTGflflflGflflflTTTGCCTflflCGflflGflTTCflflCTGflTGCTGflCTTflTCflflGflGflflTTflflTGGflCTTGfl
6120
E Y S K O P P G N N . flTGflflTflTflGTflAflGflCCCflCCCGGTflflCflflTTflflGGTGTGTflTflTGTflflTCflGGCCTGTTTGTTflGCTTGTGCRCflTTTGTTflflTTflflTflflTTTflTTTflflflflflTGGGCflflflflTflTflflflT
6210
GCflTflflflflGflTGflCflTflGRGflTflTGTGCflCflflflGCTflTGTflflTCflTflTTTflflCflTTflflTflTflTflTTflTTTCGTTflTTCflCflflTTCGTCflTGflflGGCCflGCGTflGTTTflCGGTTCCTGCTfl . L E O H L G f l Y N U T G f l
6360
CTGCATTflTCflTCGTTGflCflflTGTTTGTflTTCGflflTTflTTflTTCflflflGGTGCTGCflflTTGTTTGCGGflGGGGCCflCTGGflTTflGGCflflflGflflGTGflTCTCCflCGTTflflGTGCTGflflflTflfl U f l N O O N U l N T N S N N N L P f l f l l T O P P f l U P N P L S T l E U N L f l S l
6180
CTTCflCTflGflTflGflGflGflCGTCTTCGGTflflCflTCTflCCGTflCCRGflTGCCTGCflTTTCCGTCGTflflGCflTTCGGGflflflCflGGflGTflTTTflCflTTTTCGCTGflTTTTGTGGCGTflCCCCflfl U E S S L S U O E T U D U T G S f l O l l E T T L l l f l S U P T N U N E S l K H f l U G
6600
RCGCTTCflGflTGflflTCTTTTGflTGflCTGTflCTTCTflTCflTTflTTTCTTTCTGTflCflflflTCCTGflTGCflTCflflTTGflflCTflGCflGflTflTTTTGTGflGGGCTflCTTflCflflflflGflTGGTGCTG F f l E S S O K S S O U E l l l l E K O U F G S f l O I S S f l S I K H P S S U F S P f l
6720
TTGTGGTTRCflCCTGGCATCGGCTGflGCTflCTGTGCTTGflCGCGCTCTCCRCGGRTflRRGTTCCCTTGCCTflTflTCflGflCflGflGCGCCflflTTGflflGCflCCflCTflCTflCTflflflGflTCflTflG T T T U G P ~ P O R U T S S R S E U S L T G K G I O S L R G I S ~ G S S
S
6840 F I
O
~
~
F
E
~
K
R
~
151
SEQUENCE OF supbl-RAD 18 REGION ON CHROMOSOME 111 flTGCCflTflGflflGTCCCTTCflflTflTTTGTGGflTTCTGflTflGGGflflGflTTGflCCflRCflCTCGTCTTflGGTGflTTGTflCflGCflTCflGTGflRflflCflCTflGTGGGTTCCGflflCTGTCflGCCflflflR S f l M S T G E l N T S E S L S S O G U S T K P S O U f l O T F U S T P E S S D f l L
6960
CflCTflGTGGGTTCCGflflTTGTCflGCCflRflflCflCTflGTGGGTTCCflflflTTGTCflGCCflflCflCflCTGGTGTflTTCCGflflTTflTCflflTCflflGGCflCTGGflGCGTTCTGflflTCCTCGGTflGflGfl 7080 U S T P E S ~ D f l L U S T P E L N D f l L U S T Y E S N D l L f l S S f l E S D E T S flflGCflflTTflCTCTCGflflGTCTTCGTTTClGTTTTTGflGflflTGflGCTflGTTGCCGTCTCCflCflflTflGCTGflTGflflflCflGGTTCflGflGflTGGflflTGflflflTTTflGflflflflflGTGGflflTCTGflGfl F f l I U R S T K T E T K S F S S T f l T E U I f l S S U P E S I S H F K S F T S D
7200 S
TTGflflGGGflTTGflTTCflGflflGCflflGflflCflGflflGflflGTTflCflGTCGflTflCTTCTflTTGflCTGGflflGCCflGflGGTTflTflGflTGflflTCflGflflflflTGTflCTCGGflTGCGflGGflflflTflGflGTCCT l S P l S E S f l L U S S T U T S U E l S O F G S T I S S D S F T S P H S S l S 0
7320
GGGflTGCTGflTGTTGflCflflflflTTTCTTCflTTTGGGflTCflflflTTflGflCGCflflCTGflGTCflflflTTCGGflTGCTflTflGflflflTflGflflTGCGTflGGTGTTTCflflCCflTflCGCTCTflflTTCTGflTG Q S f l S T S L l E E N P l L N S f l U S D F E S f l I S I S H T P T E U ~ f l E L
E
CTflTGGGflTCflflTTTCGGflGflCGGGCGflflGflflflCCflTflGTGTCTGTGGGCGTCflflCflflCflTTGflflGflflGflTGTTGTflflCflTflCTGCTCGGCTTCTGCflTTGGflflTCTGflTGCflGGTflTflT ~ I P D I E S U P S S U ~ T D T P T L L ~
~
7440
S 7560 ~
TCCTflTflCTCflflTTTTTGTflflTGflCTTCflGTGflTTflTTTTTGTCTTflTflflflTCTCTCTCGTGGflTGCCGGTTTTGflGTTGTCTflGflRflflGflTGTGTTGGCTGCRTCflTTGGTGGGGTTTG N f l Y E l K T l U E T l I K T K Y I E f l T S f l P K S N O L F S T N f l f l D N T P N
7680
TTTGTGTGGTGGTflflCCGCflGTCGCGGTflCTTGTGGflTflflTGCTGTflGTflGGGCTflGTTGTTflflCGflTGflGGflflflGflGflTflflTGflflGflTGflflGflTGflflGflCGGCflTGTGTGflTflGCGflflG T ~ T T T ~ ~ T ~ T S T S L ~ T T P S T T
7800 ~
L
GTGflTTCflGTflCTTflflGflGTTTTGflflflCGCTTTCTCTflTCTGTTGTTflTTTCGCTTTCTGflTTGTTCTflCTflTTflflCGflGGflCflflTTTCTCCTCCflflGTCflGGflGTCGGCTCflGGflTflTT P S E T S L L K S U S E f l D T T l E S E S Q E U l L S S L K E E L D P T P E P Y
7920
CTGTGflTflGflflTTTGflflGflTGflGGflTflflCflflflTCTflTCGflCTCTGaa::c E T I S N S S S S L L D I S E (---YCfl9Ol
Figure 3. Nucleotide sequence of the sequenced region. The deduced amino acid sequences of the RAD18 gene and the open reading frames are indicated above or below. The arrow next to the name of the reading frames indicates the direction of transcription. The sequence of the mature serine tRNA is indicated in italics. Some restriction sites relevant to our studies are underlined.
-
YCR904
1000
2000
3000
4000
+ YCR903
YCR902
-5000
RAD18
6000
7000
aooo
YCR901
Figure 4. Identification of open reading frames with the 7965 bp fragment using DNA Strider software (Marck, 1988). Long barsare stop codons and short bars are ATG codons.
homology with published sequences. YCR902 was disrupted by insertion of the LEU2 gene between the AvaI (nucleotide 2618) and BarnHI (nucleotide 291 1) sites. This disruption was introduced by gene replacement in haploid strain MGD353-13D. The disruption was tested for the following auxotrophies: tyr, his, ile, lys, met, phe, thr and for
the growth on glycerol-complete agar plate. No differences were observed between the disrupted and control strains. RAD18 (bases 4692 to 6152) The RADI8 gene of S. cerevisiae is involved in the error-prone DNA repair. It had previously been
~
~
~
~
~
~
L
~
152
P. BENIT ET AL.
60 903 MPRIXTRRSXPAPDGFEXIXPTLTDFEIQLRDAQWXSSKLAAXSNEQLWEIMQLHHQRS
.. .. .. .. .. .. .. .. .. .. .. .. .. ..
.. .. ..
.. .. .. ..
... ...
G10 MPKVK-RSRKPPPDGWELIEPTLDELDQ~TD--PHEGKRKVESLWPIFRIHHQXT
102 903 RYIYTLYYKRKAISKDLYDWLIKEXYADXLLIAKWRKTGYEKLCCLRCIQXNETNNGSTC
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ....................................
.. .. .. ..
G10 RYIFDLFYXRXAISRELMYCIREGYADXNLIAKWKKQGYENLCCLRCIQTRDTNFGTNC 150 903 ~RVPRAQLEEEARKXGTQVSFHQCVHCGCRGCASTD
..... .....
.. ..
.. .. .. .. .. .. .. ..
G10 ICRVPXTXLEVG--------RIIECTHCGCRGCSG Figure 5.Comparison of protein YCR903 with protein GI0 of Xenopus (McGrew et al., 1989). The zincfinger domain is underlined.
sequenced by two different laboratories, including ours (Chanet et al., 1988; Jones et al., 1988; Fabre et al., 1989). The only difference found in this new determination was an additional T residue in a long stretch of T (position 4200) located between the 3’ end of YCR902 and the 5‘ end of RAD18. YCR90I (bases 6320 to 7965) This sequence is the 3’ end of an O R F that is 1065 amino acids long. The 5‘ end of this gene was sequenced by Fiers’s group (University of Gent). This protein is rich in serine and presents a repeating motif of ten amino acid residues (ADNSEPTSVL), which is repeated six times. It also contains the HDEL amino acid sequence typical of proteins associated with the endoplasmic reticulum (G. Lauquin, personal communication). The deletion of the 3’ end of this gene (the two adjacent 1607 and 2329 bp long EcoRI fragments were deleted and replaced by the LEU2 gene; Fabre et al., 1989) was not lethal in haploid cells and expressed no mutant phenotype. As judged by Northern blot analysis, the YCR901 transcript is about 3.35 kb long (F. Fabre, unpublished result). ACKNOWLEDGEMENTS This work was supported in part by the Commission of the European Communities (Biotechnology Action Program). REFERENCES Brandriss, M. C., Soll, L. and Botstein, D. (1975). Recessive lethal amber suppressors in yeast. Genetics 79,551-560.
Brandriss, M. C., Steward, J. W., Sherman, F. and Botstein, D. (1976). Substitution of serine caused by a recessive lethal suppressor in yeast. J . Mol. Biol. 102, 467476. Chanet, R., Magana-Schwencke, N. and Fabre, F. (1988). Potential DNA-binding domains in the RAD18 gene product of Saccharomyces cerevisiae. Gene 74, 543-547. Fabre, F., Magana-Schwencke, N. and Chanet, R. (1989). Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of radl8 deletion mutants. Mol. Gen. Genet. 215,425430. Jones, J. S., Weber, S . and Prakash, L. (1988). The Saccharomyces cerevisiae RAD18 gene encodes a protein that contains potential zinc finger domains for nucleic acid binding and a putative nucleotide binding sequence. Nucl. Acids Res. 16,7 1 19-7 13 1. Lin, H. C., Lei, S. P. and Wilcox, G. (1983). An improved DNA sequencing strategy. Anal. Biochem. 147, 114-1 19. Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982). Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Marck, C. (1988). DNA strider: a C program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucl. Acids Res. 16, 1829-1836. McGrew, L., Dworkin-Rastl, E., Dworkin, M. and Richter, J. (1989). Poly(A) elongation during Xenopus oocyte maturation is required for translational recruitment and is mediated by a short sequence element. Genes Dev. 3,803-8 15. Mortimer, R. K. and Hawthorne, D. C. (1973). Genetic mapping in Saccharomyces IV. Mapping of temperature-sensitive genes and use of disomic strains in localizing genes. Genetics 74,33-54. Mortimer, R. K., Schild, D., Contopoulou, C. R. and Kaus, J. A. (1989). Genetic map of Saccharomyces cerevisiae. Edition 10. Yeast 5,321403.
SEQUENCE OF sup61-RAD 18 REGION ON CHROMOSOME I11
Olson, M. V., Page, G. S., Sentenac, A., Piper, P. W., Worthington, M., Weiss, R. B. and Hall, B. D. (1981). Only one of two closely related yeast suppressor tRNA genes contains an intervening sequence. Nature 291, 464469.
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
153 strategy for genomic restriction mapping in yeast. Proc. Natl. Acad. Sci. USA 74,5463-5467. Rothstein, R. (1985). Cloning in yeast. In Glover, D. M. (Ed.), DNA Cloning. Volume II, A Practical Approach. IRL Press, New York, pp. 45-66. Sherman, F., Fink, G . and Hicks, J. (1983). Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
