Fried, H. M., Pearson, N. J., Kim, C. H., and Warner, J. R. (1981). J. Biol. Chem. 256,10176- .... Underwood, M. R., and Fried, H. M. (1990) EMBO J. 9,91-99.
Vol. 267, No. 5, Issue of February 15, pp. 3008-3013, 1992 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
The Yeast RibosomalProtein S7 and Its Genes* (Received for publication, July 15, 1991)
Dennis SynetosS, Mariana D. Dabeva, and Jonathan R. Warner6 From the Department of Cell Bwbgy, Albert Einstein College of Medicine, Bronx, New York 10461
Ribosomal protein 57 of Saccharomyces cerevisiae is encoded by twogenes RPS7A and RPS7B. The sequence of each copy was determined; their coding regions differ in only 14 nucleotides, none of which leads to changes in the amino acid sequence. The predicted protein consists of 261 amino acids, making it the largest protein of the 40 S ribosomal subunit. It is highly basic near the NH2 terminus, as are most ribosomal proteins. Protein 57 is homologous to both human andrat ribosomal protein 54. RPS7A and RPS7B contain introns of 257 and 269 nucleotides, respectively, located 11 nucleotides beyond the initiator AUG. The splicing of the introns is efficient. Either RPS7A or RPS7B will support growth. However, deletion of both genes is lethal. RPS7A maps distal to CDCl1 on chromosome X, and RPS7B maps distal to CUP1 on chromosome VIII.
Our knowledge of the structure andfunction of the eukaryotic ribosome lags far behind that of the prokaryotic ribosome. Because of its relative simplicity, its ease of handling, and itsgenetic tractability, the yeast Saccharomyces cereuisiae has proved to be a suitable eukaryotic organism in which to study ribosome structure and function (1, 2). The genes for many yeast ribosomal proteins have already been cloned and sequenced (3). As part of this process, we present here the sequences of two genes, RPS7A and RPS7B, encoding ribosomal protein S7 (formerly called RP5 (4)), the largest protein of the 40 S subunit. The open reading frames of the two copies are nearly the same and predict that RPS7A and RPS7B encode identical proteins. S7 is highly homologous to mammalian ribosomal protein S4 (5, 6). Theuntranslated sequences, upstream or downstream of the open reading frame or within the intron, diverge completely. Ribosomal protein S7 is essential for growth. MATERIALS ANDMETHODS
Strains, Media, Plasmids, and Libraries-The strain of S. cereuisiae used is the homozygous diploid W303 (Matala, ade2-1, his3-11,15,
* This work was supported by National Institutesof Health Grants GM25532 and CA13330 while D.S. was on leave from the University of Patras, Greece, and M.D.D. was on leave from the Bulgarian Academy of Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in thispaperhas been submitted to the GenBankTM/EMBL Data Bank with accessionnumber(s) M64293 (Fig. 4) and M64294 (Fig. 5). $. Permanent address: Laboratory of Biochemistry, School of Medicine, University of Patras, 261 10 Patras, Greece. § T o whom correspondence should be addressed. Tel.:212-4303022; Fax: 212-829-7619.
leu2-3,112, trpl-1, ura3-1, cad-100) obtained from R. Rothstein (Columbia University). It was grown at 30 “C in YPD (1% yeast extract, 2% peptone, 2% dextrose). Escherichia coli strain DH5a was used throughout for the propagation of plasmids. Plasmid pYERP5, containing an 11.5-kb’ EcoRI/EcoRI fragment including the RPS7A gene, was derived from a X clone (C62) as described previously (4) and was subsequently subcloned into plasmids pUC18 and pGEM-blue, and Yep24’ (7). Gene RPS7B was isolated by screening a S. cereuisiae genomic library based on the TRPl CEN3 vector M l l l (obtained from V. Mackay, Zymo Genetics, Seattle, WA).E. coli RR1, transformed with this library, was screened by transferring colonies to nitrocellulose filters (8) and probing with suitable RNA probes. From colonies giving a positive signal, DNA was isolated essentially as described by Sambrook et al. (9). Southern (DNA) and Northern (RNA) Blot Analysis-DNA digested with restriction enzymes or yeast total RNA from yeast was fractionated onagarose gels as described previously, blotted to Nytran (Schleicher & Schuell), and probed with DNA, RNA, or oligonucleotide probes as appropriate. DNA probes were prepared by nick translation according to Rigby et al. (lo),using [ Y - ~ ’ P ] ~ C T (specific P activity 3,000 Ci/mmol, Amersham Corp.). RNA probes were prepared by in vitro transcription of double-stranded DNA template with the use of T7 or SP6 DNAdependent RNA polymerase (9) in the presence of [a-32P]UTP(specific activity 800 Ci/mmol, Amersham Corp.). Synthetic oligonucleotide probes were prepared by 5’ end labeling with [c~-~’P]ATP (specific activity 3,000 Ci/mmol, Amersbam Corp.) and T 4 polynucleotide kinase (New England BioLabs) (9). Sequencing-DNA fragments of RPS7A were subcloned into pGEM-blue. The recombinant plasmid was digested with AuaI producing a 5’ overhang and with Sac1 producing a 3’ overhang and treated with exonuclease I11 using the Erase-a-Base system (Promega Biotec). The sequence of the resulting DNAs was determined by the dideoxy chain termination method using 35S-dATP according to Sanger et al. (11) applied to DNA minipreparations (12). The sequence of RPS7A was confirmed by using appropriate oligonucleotide probes. A 4.6-kb clone containing the second copy RPS7B in the plasmid MI11 was sequenced by the use of radiolabeled synthetic oligonucleotides as primers andthe dideoxy chaintermination method as described above. In all cases both strands were sequenced. Splice Site Localization-Yeast total RNA was transcribed with reverse transcriptase using as a primer a synthetic oligonucleotide complementary to the RPS7A transcript. The cDNA generated was used as a template for polymerase chain reaction (PCR),utilizing the Gene Amp DNA amplification reagent kit (Perkin-Elmer Cetus Instruments). The resulting 135-base pair PCR product was purified using the QIAGEN kit (QIAGEN Inc., Studio City, CA). The PCR product primers in the PCR reaction contained EcoRI and Hind111 linkers. Subsequent digestion with these enzymes produced a 127base pair product which was then ligated to pGEM-blue and used to transform E. coli DH5a. Plasmid DNA was isolated from individual colonies and sequenced as described above. Disruption of the S 7 Genes-The disruption of the two genes for S7 in W303 was carried out according to the method of Rothstein (13) in two steps. For disruption of RPS7B, the gene with its flanking sequences was subcloned into pBR322 after digestion of the plasmid ‘ T h e abbreviations used are: kb, kilobaseb); PCR, polymerase chain reaction.
3008
Yeast Ribosomal Protein S7 and ItsGenes isolated from the genomic library with HindIII. The subclone was cleaved with XbaI and Sac1 and the excised DNA replaced by the HIS3 gene. For disruption of the gene the plasmid was cleaved with SalI and EcoRV and used to transform W303. For disruption of RPS7A twoDNA fragments encompassing sequences outside and inside of the gene (sequences 41-1108 and 1269-2024, respectively, from the map in Fig. 3) were synthesized by PCR, using appropriate synthetic oligonucleotides. The DNA fragments were purified and cloned into pUCLEU2 in two successive steps on either end of the LEU2 gene. For disruption of the gene the plasmid was cleaved with suitable enzymes and used for transformation of W303 in which one copy of RPS7B had already been disrupted. Mapping of the S7 Genes-A X contig map of the S. cerevisiue genome and the associated X clones in a defined array of a set of nylon filters were kindly supplied by L. Riles and M. Olson (Washington University, St. Louis, MO)? The filters were probed sequentially with a subclone of RPS7A and a subclone of RPS7B which contained sequences outside the coding region to avoid cross-hybridization. L. Riles provided the information regarding the precise location of the appropriate clones. The chromosome identity was confirmed by probing a chromosome blot.
mkb
A
I
E
H
B
Cloning RPS7A"The gene for ribosomal protein S7 was originally cloned as an 11.5-kb EcoRI fragment in a X vector (4). The product of the genewas identified as ribosomal protein S7 (formerly called RP5) by in uitro translation of mRNA hybridizing to the DNA fragment and subsequent two-dimensional polyacrylamide gel analysis (4). Portions of the 11.5-kb EcoRI fragment were then subcloned in pGEMblue vectors. One of these subclones contained the 2.2-kb BglIIIBglII fragment which includes the gene for the ribosomal protein S7 (Fig. 1).The location of the gene and the orientation of its transcript were determined by Northern analysis of yeast total RNA, using appropriate RNA probes transcribed from this clone. Cloning RPS7B-A Southern blot of yeast genomic DNA digested either with SalI or EcoRI was probed with a nicktranslated DNA probe from outside the coding region of RPS7A (Fig. 2 A ) . In either case only one band was detected. When the same blot was hybridizedwith a probe from within the coding region, twobands were detected in each case (Fig. 2B), suggesting that S7is encoded by two genes. The second, RPS7B, was subsequently isolated by screening a genomic library with the use of an RNA probe transcribed from within the coding region of RPS7A (see "Materials and Methods"). From about 16,000 coloniesscreened, 30 gavea positive signal. Twelve of these colonies were analyzed, of which two were putatively identified as carrying RPS7B since they gave a signal with a probe of coding sequences but notwith noncoding sequences of RPS7A. Fig. 1B shows a partial restriction map of the genomic clonecontaining RPS7B, from which the location and direction of transcription are deduced. Sequencing RPS7A and RPS7B"The sequence of RPS7A is shown in Fig. 3,and thatof RPS7B in Fig. 4. Each sequence contains two diagnostic signs of an intron: a 5' splice site (GTATGT) and a lariat site (TACTAAC). Confirmation of the splice site of RPS7A is described below. Splicing of this intron yields an open reading frame of 261 codons in each gene. The coding regionsof the two genesare nearly identical, differing in only 14 nucleotides out of783, resulting in no change of amino acids. On the other hand, the two genes display virtually no sequence homology in their introns or in their 5'- and 3'-noncoding regions. The sequences of RPS7A upstream of the open reading frame are characteristic of a ribosomal protein gene. There
* L. Riles and M. Olson, personal communication.
E
HE H
5'
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0.4 kb
i
SS
1.0 kb
: S
RESULTSANDDISCUSSION
3009
.
1..
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FIG. 1. A, restriction map of the 11.5-kbEcoRI fragment that contains RPS7A and subcloning scheme. Sequencing of the 2.2-kb BglII/BglII subclone was carried out as described under "Materials and Methods." The transcript (lower line) consists of exons 1 and 2 (solid bars), 14 and 769 nucleotides long, interrupted by an intron (open bar), 257 nucleotides long. B, restriction map of the 4.6-kb Sau3Al fragment that contains RPS7B. A 1.17-kb portion that includes an SalI DruI fragment and contains RPS7B was sequenced initially using oligonucleotides from RPS7A as primers. After part of the sequence of RPS7B was deduced, oligonucleotides from RPS7B were used as primers to complete the sequencing. The transcript (lower line) consists of exons 1 and 2 (solid bars), 14 and 769 nucleotides long, interrupted by an intron (open bar),269 nucleotides long. Restriction site: B, BglIk D, DraI; E, EcoRI; H, HindIII; P, PvuII; S, Salk S I , Ss Sau3Al; X , XbaI.
B.
A.
kb 23.19.46.6 4.4-
-
- -
..
.
w" ' I
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11 22 33
FIG. 2. Copy number analysis of genes for protein 57. A Southern blot of yeast genomic DNA digested with either Sun or EcoRI was hybridized to an RNA probe transcribed either from the noncoding region of RPS7A ( A ) or from the coding region of RPS7A ( B ) .Bands a t 20 kb for SalI and 11.5 kb for EcoRI in the two panels represent gene RPS7A. Bands a t 12.5 kb for SalI and 4.0 kb for EcoRI, in B, indicate the existence of a second copyof the gene encoding protein S7, RPS7B.
are four potential TATA boxes at positions -98, -86, -81, and -48, a T-richregion from-278 to -340, a HOMOLl box at -349 to -360, and finally an RPG box at -374 to -385. HOMOLl and RPG each bind the RAP1 protein and have beenshown to be upstream activating sequences for the transcription of genes for ribosomal proteins, transcription factors, and RNA polymerase subunits (14-17). Downstream of RPS7A (1287-1316) is found an interesting run of 15 consecutive GTs, with the potential to form Z DNA (18).
Yeast Ribosomal Protein 5'7 and Its Genes
3010
-447 CATCTMTCCTTCTCCTGGCCTACCGTCTGTGCMCCATTAGTCATCATTACGTGTGTT~~CTTATCTTGT~TTAT~~TACTTTTCGTAGTC -547
CTTTCCTGCGUCGTTGTCUTTTTCGTMCUCGT~GTCCTMTGCACAGCT~GTACT~TTTTTTCCATGTTTT~TATT~GTT~~G
-447
G M A T M A C M A T M A C M A U T T A T A T C T G G T T T T T T T T C C U C T
-347
TCTGTMTTTTGCTTCCTTTTTCTTCCTGCGTTCTTTTTTTCTTGMCTGTCGTTTTCCGTTATTTTTTTCGGTGA~T~GTT~GTA~~GGCC
-247
TAGGCCACGGTAGCTCTTTGTAGTCGTGGTMGGGGCAGTA~TT~CTTAGTACGTGGTCTTGCAGTTAGGCTG~TC~CTGGCCCT~CMGTC
-147
CTGTTCTGTGTGGTAGTATTGAMTTTCACACATTGTCCGTACTAGTATATT~TTATACTATMTTTMTCTAGTGTTGMATACTTTCTTAT
-47 1
ATAGCCATTTTTCTGCCC~CCMAGMTCMTACG~GATGGCTAGAGCACC~TTCACTATACACTTTCATTATMTTAC~GG
M
A
R
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54 A T C A G M G M T C A T A C A C M G M A C M G T G G A G T C T T M C ~ C G M T A G G M C M C M T ~ C ~ G T T T A T G T C ~ T T T M T T T T A C A T ~ T C C T ~ G 154 A T T G T A N W T A T T T T A C C A G T M T C A T T ~ C A G ~ C M T ~ T A T G T C T G T A T C T T ~ T T A T A ~ T T U T T T T T ~ C C T T T T T T C 254 6
TUTTTTTTTCCGTAtACUAGMGCATCT~CATTAGCAGCTC~~CCACTGGTTATTGCACMGTTGTCCGGTTGTTACGCCCCMCACUTCT K K H L K R L A A P H H Y L L D K L S G C Y A P R P S
354
GClGGTCWCICMATlGCGTGMTCCTTGCCATTWTTGTCTTTCTM~CACATTMAGTATGCTTT~CGGCCGT~GTCMGGCTATCTTW
3
3
A
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K
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454 T G C M C G T U C G T T M A G T G G A C G G T M G G T T A G M C C C A ~ C T A C C T A C C ~ G C T G G T T T ~ T G C A T G T C A T C A C T C T A C A T ~ ~ C C M T ~ C T T 67 ~ R H V K ~ ~ G K V R ~ D ~ ~ Y P A G F ~ ~ V I T L
~
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554 C A G A T T G G T C T A C C A T G T U G G G T A C A T T C G C T G T C ~ C C G T A T ~ C C G A T G M ~ G C T T C T T A C M G T T G G G T M G G T C M G M G G T T C M T T A G G T 100 R L V Y D V K C R F A V H R I T D E E A S Y K L G K V K K V ~ L G 654 M G M G G G T G T T C U T A C G T T G T T A C C C A C G A T G C T A G M C T A C T T G G C C T 1
3
3
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754 CTGGTMCATTACTCATTTUTCMGTTCGATGCCGGTMGTTGGTTTACGTTACTGGTGGTCGTMCTTGGGTCGTATCGGTACTATCGTTUCMGCA 167 G K I T D F I K F D A G K L V Y V T G G R N L G R I G T I V H
A
S
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a54 MCAUCGATGGTGGTTTCWTTTAGTT~~TCMGGACTCCTTGGACM~CTTTCGT~CTACATT~CMTGTCTTCGTUTCGGTGMCMGGT 200 R H D G G F D L V H I K D S L D W T F V T R L N N V F V I G E
E
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954 MGCCTTAUTTTCTTTGCCMAGGGTMGGGTATCMGTTGTCTATTGCTGMGMCGTCA~GMGMCAGCTCMCMGGTTTATMATTT~TMC ~
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1054 M C T T M T T A T T T T C T T C T T T T G T A T A T C T C ~ T T M T G T T T A T T A ~ T T G M T T T T ~ T M T A U T C G T A T C T T C C T T T T T C C A C T G G ~ G T M T 1154 A T M C G T A T M T A T A T A T A T T A G G T G T G T G T A T A T A T A T C C G T A T T G T M T A T T C A T A G T ~ T A C G C T M C C C T ~ T A G M ~ C G T A T C A T M C A 1254 CGTACTWCAETACGWCTACCACMTATAT~GTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCATTGTATTGGMTATATATACTTACTATTA 1354 AGCTTATATGGTTCGCATATTCACTATTTATMGGATATTCMCTTGTATGTCCTTTCTTMCCMATTTTCTTCTTTCTCTTGGTGGTM~TGTTC~ 1454 C M A C T T C T C A G T A C M l G A T C C C T T T G M T T T C T T T A T ~ C A G G G T C C ~ T M T T C A G M C C C A C G C C C A C A T C
FIG. 3. Sequences of RPS7A and predicted aminoacid sequence. The upper line represents the complete nucleotide sequence of RPS7A. All sequence was determined on both strands. The lower line corresponds to the predicted amino acid sequence of both open reading frames. The consensus sequence of the intron as well as the HOMOLl and RPG boxes at -349 and -374 nucleotides are underlined.
Identifitation of the Splice Sites of RPS7A"To confirm the position of the splice sites, the PCR strategy shown in Fig. 5 was followed. Yeast total RNA was used as a template for reverse transcription, employing as a primer an antisense oligonucleotide downstream of the putative introns (oligonucleotide 1, Fig. 5A (+380 + +361)). The resulting cDNA was used as a template in aseries of PCR reactions. Oligonucleotides 2 and 3 (Fig. 5A (-7 4 +13 and -71 + -49, respectively)), lying upstream of the 5' end of the coding region, were used as sense primers. Only the reaction using oligonucleotide 2 generated a PCR productof the size expected from a spliced mRNA (Fig. 5 B ) . We conclude that transcription initiates between positions -60 and -8. From the sequence of the PCR product using oligonucleo-
-
tide 2 (Fig. 5C) it is clear that the 5' splice site is C:GT and that the 3' splice site occurs 81 nucleotides downstream of the TACTAAC box, immediately following the second AG. Although in most cases the first AG is used, in this case the first AG is only three nucleotides from the lariatsite. Forthe predicted intron of RPS7B, the 3' splice site occurs 40 nucleotides downstream of the lariat site, immediately following the third AG. The single band detected by PCR (Fig. 5 B ) suggests that splicing of this intron is efficient. To confirm this and toask whether S7, like ribosomal protein L32 (19, 20), might regulate. the splicing of its own transcript, Northernanalysis was carried out on RNA from yeast cells carrying RPS7A on a multicopy plasmid (Fig. 6). Lane 3 of Fig. 6, representing a
E
N
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Ribosomal Yeast Protein
- 74
Genes S7 Its and
3011
GTMWTTTAGMTACTTTCTTTTUTATMCGTCGACTMGTATMUATAGATACACCACTATTGAGGMACATGGCTACAGGACC~TGATTT M A R G P
1
27 C W C C T ~ T C M C A G T T C C C M M C M G A T M T A G T T T T C T T T ~ G A T G G G T A C C C T C T C A T G A T T G G T A U A G T G A T T T G U C ~ G T G A C G 127 A T C C G G A C T M A G M A C M T A T M ~ G T T G T G T T T A T C T A T C G C U C A T A ~ T T C T C A T C A ~ C T T T A T C C T T G T T M ~ U G A T M G C A T T G C 227 6
GGGATATTTT~MCAGTACGTTTMTMTGTTMTACCATTTTTUTATAGMA~GUTCTMACACATTAGUGCTCCACACCATTGGTTA K K H L K R L A A P H H U L
327 20
TTCGAUAGTTCTCCCGTTCTTACGCCCCMGACUTCTGCTGGTCCAU~TTGCGT~TCCTTGCCATTGATTGTCTTTCTMGMAUCATTM L
D
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427 AGTATGETTTCUCCCCCCTCMCTCAIGCTATCTTGATGCMCGTCACGT~GTTGACGGTMGGTTA~CTGAUCCACCTACCCAGCTGGTTT Y A L W C R E V W A ~ L ~ P R ~ V K V O G K V R ~ D ~ ~ 54
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527 U T G G A C G T U T U C T C T A C A T G C U C C M T ~ C T T C A C A T T G G T C T A C G A T G T C M G G G T A G A T T C G C T G T C C A C C G T A T U C C C A T ~ ~ G C C 87 M O V I T L D A T N E W F R L V Y O V K G R F A V H R f T D E E A 627 TCTTACMATTCCGTMGGTCMCUGGTTCMTTAGGTMCMGGGTGTTCCATACGTTGTTACCCACCATGGTACUCTATCACATACCCACACCCM 120 S Y K L G K V K K V P L C K K G V P Y V V ~ H D G R ~ I R Y P
~
P
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727 ACATCMCGTCMTCACACTGTTMGATTCATTTGGCCTCTGGTMCATTACTGATTTCATCMGTTCGATGCCGGTMGTTGGTTTACGTTACTGGTGG I K V W D T V K I O L A S C K I T O F I K F O A G K L V Y V T G G 154 a27 T C C T M C T T G G G T C C T A T C C C T A C T A T C G T T C A C M G ~ ~ C A C ~ ~ G G ~ G G ~ ~ ~ C G A T T T G G T T C A C A T C M G G A C T C C T T G G A C M C A C T T T C G T C 187 R W L G R I C T I V H K E R H O C G F D L V H I K O S L O ~ T F V
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ACTAWTTCMCMTGTCTTCGTCATTCGT~CMGGTMGCC~~ACA~~~C~TTGCCMAGGGTMGGGTATCMGTTGTCTATTGCTCU~CGTG
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1027 ACACMGMWGCTCMCMGCTTTGTMACATTTTMATATTGTTATCTGCCCTCTCTTCGTCTTTTG 254 R R R A P P G L '
FIG. 4. Sequence of RPS7B and predicted aminoacid sequence. See the legend to Fig. 3.
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FIG. 5. Determination of RPS7A splice sites.A, schematic representation of the method. A PCR product will be obtained only when the mRNA from which the cDNA is synthesized contains thesequence complementary to the upstream oligonucleotide (oligonucleotide 2). When transcription starts downstream of the oligonucleotide (oligonucleotide 3 ) , no product will be obtained. B, oligonucleotides 1 and 2 with no RNA in the initial reverse transcription reaction (control; lanes I and 2 ) ,oligonucleotides 1 and 3 (lanes 3 and 4 ) , and, finally, oligonucleotides 1 and 2 (lanes 5 and 6 ) . Only the last combination gave a PCR product. C, sequencing gel showing the splice site indicated by an arrow.
prp2 mutant (21), has an upper band corresponding to precursor mRNA, caused by defective splicing at the restrictive temperature.Nosuchband is detectedeither for control strains (lanes 1 and 2 ) or for a strain overexpressing S 7 (lane 4 ) . Thus the intron of the RPS7A transcript is effectively spliced, even when overexpressed. Characteristics of Ribosomal Protein S7"The ribosomal protein S7 is a basic protein of 261 amino acids. The ratio of the basic to acidic amino acid residues is 47:27 for the entire molecule but 9:l for the first39 amino acid residues. The NH2 terminus of ribosomal protein S7 is identical to the NH, terminus of the ribosomal protein termed YS6 (22), confirming the proposed identity of these two proteins(23). Compar-
ison with the nuclear localization signal of SV40 T antigen (24) and with ribosomal protein L29 (25) suggests that amino acids 6-12, PKKHLKR, may be the nuclear localization signal of S7. As with other ribosomal protein genes, the codon usage of both RPS7A and RPS7B shares the biasfound in genesfor other abundant yeast proteins(26, 27). S7 Is an Essential Protein"RPS7A was disrupted by insertion of the LEU2 gene with the concomitant removal of 260 nucleotides from the coding region. RPS7B was disrupted by the insertionof the HIS3 gene with the concomitantremoval of 498 nucleotides from the coding region. The two disrupted genes were used in stepwise fashion to replace the wild-type genes in a diploid cell by the method of Rothstein (13).The
Yeast Ribosomal Protein
3012
1
2
3
GenesIts S7 and diploid was sporulated and asci dissected. Fig. 7 shows a Southern analysis of the three live spores of a tetrad,with the phenotypes Leu'His-, Leu-His-, and Leu-His+, together with the wild type. No Leu+His+spores were obtained, suggesting that a cell with no genes for S7 is inviable. We conclude that S7 is an essential protein. However, His+Leu- and Leu+Hisspores were found at the expected frequency. The resulting cells grow somewhat more slowlythan wild type. Thus, either RPS7A or RPS7B can support growth. Genetic LocalizationofRPS7A and RPS7B"Probes specific for each gene were used to probe an ordered X bank of S. cerevisiae DNA? RPS7A hybridized to clone 5167; which is located on the right arm of chromosome X, distal to CDCl1. RPS7B hybridized to clone 3427,3 which is located on the right arm of chromosome VIII, distal to CUPl. The chromosome identifications were confirmed by hybridizing the genes to chromosome blots. Yeast S 7 Is Homologow to Mammalian S4-A search of the GenBank revealed that yeast RPS7A and RPS7B are highly homologous tothe mammalian ribosomal protein S4, sequenced in both thehuman (6) and the rat (5)(Fig. 8).Human S4 is encoded by genes on the X and the Y chromosomes whichyield slightly different proteins (6). 188 of the 261 amino acid residues of yeast S7 are identical to rat S4 and human S4X, and 183 to thehuman S4Y. The mammalian S4 is 2 amino acid residues longer than yeast S7. Fisher et al. (6) have suggestedthat haploinsufficiency of S4 may bethe cause of the human disease, Turner's syndrome. The availability of a yeast homologue may prove useful in the analysis of this complex phenotype.
4
FIG.6. Splicing of RPS7 transcripts. Total RNA from wildtype yeast cells (lune I ) , total RNA from yeast cells carrying the multicopy plasmid Yep24' (lune 2), poly(A+) RNA prepared from yeast cells with a ts mutation in the gene prp2 leading to defective splicing at therestrictive temperature (lune 3), andfinally, total RNA from yeast cells carrying RPS7A on Yep24' (lane 4 ) . All four lunes were hybridized to a ["'PIRNA probe transcribed from within the coding region of RPS7A.
1
S7R
'
2
3
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1
S7A'
S7R'
S7B'
8
S7B
Acknowledgments-We thank L. Riles and M. Olson for the X bank, advice, and information. FIG. 7. Southern analysis of spores carrying disrupted alleles of RPS7 genes. A diploid with the genotype RPS7AI rps7a::LEU2, RPS7B/rps7b::HIS3 was sporulated and a tetrad dissected. Only three spores, of phenotype Leu+His- (lane I ) , Leu-His(lune 2), Leu-His+ (lune 3) grew.DNAwas prepared from these strains and from the wild-type parent (lune 4 ) , cut with EcoRI, electrophoresed, blotted, and probed with a nick-translated plasmid that included the entire RPS7A gene. This probe detects the coding sequences of RPS7B, many of which have been deleted in the generation of rps7b::HIS3, leading to thelight band in lane 3. The positions of the intact and the disrupted (indicated by *) RPS7 genes are indicated. The size of the EcoRI fragment carrying the RPS7A gene is 11.5 kg; that carrying the RPS7B gene is 4 kb. Note that the LEU2 gene has an EcoRI site; thus the disrupted RPS7A gene is found in two EcoRI fragments.
REFERENCES 1. Warner, J. R. (1989) Microbiol. Rev. 53, 256-271
2. Warner, J. R., Baronas-Lowell, D. M., Eng, F. J., Johnson, S. P., Ju, Q., and Morrow, B. E. (1990) in The Ribosome: Structure, Function and Evolution (Hill, W. E., Dahlberg, A., Garrett, R. A., Moore, P. B., Schlessinger, D., and Warner, J. R., eds) pp. 443-451, American Society for Microbiology, Wash. D. C. 3. Lee, J. C. (1990) in The Yeasts (Rose, A. H., and Harrison, R., eds) Ed 2, Vol. 4, pp. 489-539, Academic Press, New York 4. Fried, H. M., Pearson, N. J., Kim, C. H., and Warner, J. R. (1981) J.Biol. Chem. 256,10176-10183 5. Devi,K.R. G., Chan, Y-L., and Wool, I. T. (1989)Biochem. These clones are available from the ATCC.
yeast 27 RARGPKKHLK R W H H U L L DKLSCCVAPR PSACPHKLRE SLPLIVFLRY RLKVALYGRE VKAILWQRHV KWCWRTDT TVPAGFIOVI rat Y .V...K..M. T.VF... ..T C I T.D. K.C...FI .I I
..........
hunsn YV
.......... .V...K..M.
...
...T.VF...
..T
....... .... .......... ....... .... .......... C
V
T.D.
.. ..K.C...FI
....... ..........
.I
.....V.V ..........
90 90 90
yeast S7 TLDATNENFRLWDVKCRFAVHRITOEEASYKLCWKKVPLCKKGWYWTHDGRTIRVPDPYIKVNDTVKIDLASCKITDFIKFDACKL180 r a t S4 SI.K.G.... .I..T..... P...K C..R.IF V.T..I.HL. A...... L..I &..ET.... hunnn Y Y S1EK.C.H.. .V..T..... V...K C . J . 1 7 V.V..I.HL. A...... V V P...GT...I W
180
yeast ST VnnCCRYLC RICTIVHKER HDCCFDLVHI KDSLDWTFVI RLYYVFVIGE PCKPVISLPK GKCIKLSIAE ERORRRAWC L r a t S4 a....A... V.TWR.. .P.S..V..V ..AYG.S.A. S.I....K GY..U....R hunsn 541 W.I..A... .V.V.TNR.. .P.S..V..V ..AYG.S.A. S.I....K CY..U....R
261 263 263
.....
.....
...
...
...
...
...
.. ..
..
......T.N. .....1. N.
.. ......
....R.T... ... .... ...
SSC K.L.TKP SSG K.L.AKP
R.TV..
180
FIG. 8. Amino acid sequence homology between S. cerevisiae 57,rat 54, and human S4X and S4Y. Human S4X is identical to rat S4. Identical amino acid residues are indicated by a dot. These proteins also show some homology to thesmall subunit C proteinof the archaebacterium, Methanococcus vannielii (28).
Yeast Ribosomal Protein S7 and Its Genes Biophys. Acta 1 0 0 8 , 258-262 6. Fisher, E. M.C., Beer-Romero, P., Brown, L. G., Ridley, A., McNeil, J. A., Lawrence, J. B., Willard, H. F., Bieber, F. R., and Page, D. C. (1990) Cell 63,1205-1218 7. Pearson, N. J., Fried, H. M., and Warner, J. R. (1982) Cell 2 9 , 347-355 8. Grunstein, M., and Hogness, D. S. (1975) Proc. Natl. Acud. Sci. U. S. A. 72,3961-3965 9. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Ed 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 10. Rigby, P. W. J., Dieckman, M., Rhodes, C., and Berg, P. (1977) J. Mol. Biol. 113, 237-251 11. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc.Natl. Acud. Sci. U. S. A. 74,5463-5467 12. Kraft, R., Tardiff, J., Krauter, K. S., and Leinwand, L. A. (1988) Bwtechniques 6 , 544-547 13. Rothstein, R. (1983) Methods Enzymol. 1 0 1 , 202-211 14. Teem, J. L., Abovich, N., Kaufer, N. F., Schwindinger, W. F., Warner, J. R., Levy, A., Woolford, J. L., Leer, R. J., van Raamsdonk-Duin, M.M.C., Mager, W. H., Planta, R. J., Schultz, L., Friesen, J. D., Fried, H., and Rosbash, M. (1984) Nucleic Acids Res. 12,8295-8312 15. Rotenberg, M. O., and Woolford, J. L., Jr. (1986) Mol. Cell. Biol. 6,674-687 16. Leer, R. J., van Raamsdonk-Duin, M.M., Mager, W. H., and
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Planta, R. J. (1985)Genet. Curr. 9 , 273-277 17. Huet, J., Cottrelle, P., Cool, M., Vignais,M. L., Thiele, D., Marck, C., Buhler, J. M., Sentenac, A., and Fromageot, P. (1985) EMBO J. 4,3539-3547 18. Rich, A., Nordheim, A., and Wang, A. H.-J. (1984) Annu. Reu. Biochen. 6 3 , 791-816 19. Dabeva, M.D., Post-Beittenmiller, M. A., and Warner, J. R. (1986) Proc. Natl. Acud, Sci. U. S. A. 8 3 , 5854-5857 20. Eng, F. J., and Warner, J. R. (1991) Cell 65, 797-804 21. Rosbash, M., Harris, P. K.W., Woolford, J., and Teem, J. L. (1981) Cell 24,679-686 22. Otaka, E., Higo, K., andOsawa, S. (1982) Biochenistry21,45454550 23. Warner, J. R. (1982) in The Molecular Biology of theYeast Saccharomyces (Strathern, J., Jones, E., and Broach, J. R., eds) pp. 529-560, Cold SpringHarbor Laboratory, Cold Spring Harbor, NY 24. Kalderon, D., Richardson, W. D., Markham, A. F., and Smith, A. E. (1984) Nature 3 1 1,33-38 25. Underwood,M.R., and Fried, H. M. (1990) EMBO J. 9,91-99 26. Bennetzen, J. L., and Hall, B. D. (1982) J. Biol. Chem. 2 5 7 , 3026-3031 27. Anderson, S. G. E., and Kurland, C. G. (1990) Microbiol. Reu. 54,198-210 28. Auer, J., Spicker, G., and Bock, A. (1989) J.Mol. Biol. 2 0 9 , 2136
