MOLECULAR AND CELLULAR BIOLOGY, Apr. 1992, p. 1680-1686 Vol. 12, No. 40270-7306/92/041680-07$02.00/0Copyright © 1992, American Society for Microbiology

Fine-Structure Map of the Human RibosomalProtein Gene RPS14t

JEAN-JACQUES DIAZt AND DONALD J. ROUFA*Center for Basic Cancer Research, Division of Biology,Kansas State University, Manhattan, Kansas 66506

Received 29 July 1991/Accepted 16 January 1992

We have used polymerase chain reaction-mediated chemical mutagenesis (J.-J. Diaz, D. D. Rhoads, andD. J. Roufa, BioTechniques 11:204-211, 1991) to analyze the genetic fine structure of a human ribosomalprotein gene, RPS14. Eighty-three DNA clones containing 158 random single-base substitution mutations wereisolated. Mutant RPS14 alleles were tested for biological activity by transfection into cultured Chinese hamstercells. The resulting data permitted us to construct a map of the S14-coding sequence that is comparable toavailable fine-structure genetic maps of many prokaryotic and lower eukaryotic gene loci. As predicted fromthe multiplicity of protein-protein and protein-RNA interactions required for ribosomal protein transport andassembly into functional ribosomal subunits, the distribution of null mutations indicated that S14 is composedof multiple, functionally distinct polypeptide domains. Two of the protein's internal domains, designateddomains B and D, were essential for S14 biological activity. In contrast, mutations which altered or deletedS14's amino-terminal 20 amino acid residues (domain A) had no observable effect on the protein's assembly andfunction in mammalian ribosomes. Interestingly, S14 structural domains deduced by in vitro mutagenesiscorrelate well with the RPS14 gene's exon boundaries.

Genes encoding the 40S ribosomal subunit protein S14(RPS14) have been isolated from human (21) and Chinesehamster (24) DNAs. Both mammalian S14 loci are composedof five exons whose protein-coding sequences are >91%identical. Indeed, the two genes encode exactly the same151-amino-acid polypeptide (21, 22). In contrast, althoughhuman and hamster RPS14 introns are located at identicalsites within the S14-coding sequence, they differ dramati-cally with respect to their lengths and base sequences (24).Nonetheless, human RPS14 is fully functional when trans-fected into cultured CHO cells (23).Whereas some information regarding transcriptional reg-

ulation of mammalian RPS14 is available (18, 19, 23), virtu-ally nothing is known about the functional significance ofamino acid sequence motifs within the S14 protein itself.Ribosomal proteins (r-proteins) have been described asmultifunctional polypeptides, as they must (i) be efficientlytransported to the cell's nucleolus, (ii) be accurately assem-bled into nascent 40S and 60S ribosomal subunits, and (iii)support the ribosome's participation in protein biosynthesis.Because each of these activities is believed to depend uponmultiple protein-protein and protein-RNA interactions, apriori it was reasonable to expect that r-protein S14's pri-mary structure might be composed of several distinct func-tional domains.

Mutational genetics offers a powerful experimental ap-proach for investigating functionally significant aspects of aprotein's structure. However, with conventional techniques,the approach is most suitable for prokaryotic and lowereukaryotic organisms in which high-resolution genetic meth-ods have been established. Fortunately, recombinant DNA

* Corresponding author. Electronic mail address: DROUFA@MATr.KSU.KSU.EDU.

t Contribution no. 92-56-J from the Kansas Agricultural Experi-ment Station.

t Present address: Laboratoire de Biologie Moleculaire et Cellu-laire, Faculte de Medecine Alexis Carrel, Lyon, France.

technologies provide alternative molecular approaches toanalyze protein-coding DNA sequences cloned from organ-isms not amenable to fine-structure analysis by recombina-tional genetics. In a previous report, we described tissueculture experiments designed to isolate nonfunctional (i.e.,null) mutations in the human RPS14 locus (6). Although thetissue culture selection scheme used was successful, unan-ticipated somatic recombination and gene conversion eventsprecluded the isolation of a comprehensive set of RPS14missense mutations. In addition, selection in tissue culturedid not permit us to recognize functional missense mutationsin the S14-coding sequence. Rather, it yielded exclusivelyRPS14 null alleles that harbor missense and nonsense muta-tions. To overcome the experimental limitations imposed bygenetic selection in tissue culture (6) and to mark the humanRPS14 protein-coding sequence with a diverse array ofmissense mutations, we undertook the in vitro genetic anal-ysis described in this report.

MATERIALS AND METHODS

Enzymes, reagents, and cell lines. Commercial sources oftissue culture reagents as well as restriction endonucleasesand other enzymes used in these experiments have beendescribed elsewhere (2, 6, 7, 21, 23, 24). All enzymes wereused according to instructions provided by the suppliers.Radioactive [a-32P]dCTP (800 Ci/mmol), [a-32P]UTP (400Ci/mmol), and [35SJmethionine (1,153 Ci/mmol) were pur-chased from New England Nuclear Corp. Synthetic oligo-nucleotides were produced by The Midland Certified Re-agent Co., Midland, Tex. DNA constructions were carriedout in the plasmid vector pUC13 (29), using Escherichia coliTBi (Bethesda Research Laboratories, Inc.) as the bacterialhost. For cell-free transcription, S14-coding sequences wereintroduced into the plasmid vector pGEM-1 and subclonedin E. coli C600 VCS, both of which were obtained fromStratagene, Inc. Emr-2-2 (13) is an emetine-resistant (emtb)mutant clone of CHO cells that harbors two recessive poirt

1680

FINE-STRUCTURE MAP OF RPS14 1681

c d_0. , ,lu

bB. 225bp

a i

i

_1 '_ --S I Hpa I

Tradion

r

P3I

P4

I slil SOP 1 XhoIHd III

FIG. 1. In vitro mutagenesis strategy for the human r-proteinS14-coding sequence. A minimal expression gene encoding humanRPS14 was modified from the plasmid clone pCS14-83 (19) and usedas a duplex target DNA for PCR-mediated nitrous acid mutagenesis(7). S14 exons are indicated as stippled boxes labeled El (exon I) andEII-EV (fused exons II to V), noncoding flanking and interveningDNAs are represented by thin lines, and plasmid vector sequencesare indicated by hatched boxes. As indicated by the arrow, the S14protein-coding sequence initiates 3 bp downstream from the begin-ning of exon 11 (21). Four oligonucleotide primers, P1 to P4 (Table1), were used to PCR amplify the mutagenized DNA as threeoverlapping fragments labeled A, B, and C. Restriction fragments a

to d, which together comprise the entire S14-coding sequence, were

cleaved from amplified DNAs and subcloned into a modified humanS14 expression vector, pCS14-140. Reconstituted S14 expressiongenes therefore contained mutations in defined segments of ther-protein-coding sequence.

mutations affecting the 3' end of the RPS14 protein-codingsequence: Arg-149--*Cys and Arg-150---His (22).PCR-mediated chemical mutagenesis. A plasmid DNA

clone encoding human r-protein S14 was mutagenized withnitrous acid as described previously (7). To maximize theyield of base substitution mutations evenly distributedthroughout the S14-coding sequence, we constructed a con-

venient mutagenesis target DNA and minimal S14 expres-sion vector (pCS14-140) from the human RPS14 DNA clone,pCS14-83 (19). The modified target DNA contained a

1,797-bp deletion (between SmaI and HpaI restriction sites)within RPS14 intron 1 and a 417-bp deletion (between XhoIIand HindIII sites) in the downstream chromosomal DNAsequence (21). These deletions permitted us to use an exon Ioligonucleotide (P1; Fig. 1) and the pUC13 antisense DNAprimer (P4; Fig. 1) to amplify the 5' and 3' ends of theS14-coding sequence by means of the polymerase chainreaction (PCR). The target DNA used, therefore, contained32 bp of upstream flanking sequence, human RPS14 exon I(54 bp), a short region of intron 1 (87 bp), fused exons II toV (501 bp), and 99 bp of downstream flanking DNA (Fig. 1).Construction of pCS14-140 involved destroying the NdeIand SspI cleavage sites within the vector sequence (original-ly derived from pUC13) by standard gap-filling and linkerinsertion methods. This treatment rendered the NdeI andSspI sites within pCS14-140 coding sequences unique (Fig.1) and facilitated assembly of mutant S14 expression clonesfor biological testing.The S14 target DNA was mutagenized with 1 M nitrous

acid at 22°C for 10 to 120 min. After treatment, the DNAswere PCR amplified as three overlapping nucleic acid frag-ments (A to C; Fig. 1), using four synthetic DNA primers (P1to P4; Fig. 1 and Table 1), as described previously (7).S14-coding regions (labeled a to d in Fig. 1) were excisedfrom the amplified DNAs by cleavage at flanking restrictionsites. Region a was an 86-bp HpaI-Bsu36I DNA fragmentderived from PCR product A, region b (157 bp) was a

TABLE 1. Oligonucleotide primers used for PCR amplificationand DNA sequence analysis of r-protein S14-coding sequences

Primer Source DNA sequence

P1 Exon I, sense strand 5'-AGTCTGGAGACGACOGT-3'P2 Exon II, sense strand 5'-GGTCATCAGCCTCGGACCTCA-3'P3 Exon III, antisense strand 5'-CAGCTCCTTGCACCTCTGGGC-3'P4 pUC13, antisense strand 5'-CACAGGAAACAGCTATGACC-3'

Bsu36I-NdeI fragment excised from PCR product B, andregions c (127 bp) and d (133 bp) were NdeI-SfiI andSfiI-SspI DNA fragments purified from PCR product C.Mutagenized DNA segments (a to d) were subcloned intopCS14-140 to generate four expression libraries, each ofwhich harbored nitrous acid-induced point mutations withina different region of the human S14-coding sequence.

Structural and functional analysis of mutant DNAs. Muta-genized human S14 DNA clones were screened for single-base alterations by DNA sequence analysis (4, 7, 21, 22, 25)using primer oligonucleotides P1 to P4 (Table 1) and for theirabilities to functionally complement a recessive emetineresistance RPS14 mutation carried in the Emr-2-2 line ofCHO cells (6, 23). Accordingly, we transfected individualmutant S14 DNAs cloned in pCS14-140 into Emr-2-2 cellstogether with the selectable plasmid, pSV2Neo (26), andscored G418-resistant transformed colonies for their sensi-tivities to emetine. Under the test conditions used (seebelow), biologically active S14 DNAs rendered 80 to 85% ofthe transformants sensitive to emetine, whereas inactiveDNAs (or DNAs that encode new emtb alleles) did not affectEmr-2-2 cells' drug resistance phenotype.

Transfection assays were carried out by using the Poly-brene-dimethyl sulfoxide procedure (15, 23, 24). Emr-2-2cells (7 x 10 ) were transfected with a mixture of a clonedmutant S14 DNA (7 ,ug) and pSV2Neo (0.5 ,ug). Under theseconditions, the molar ratio of pCS14-140 clones (4,220 bp) topSV2Neo (5,825 bp) was approximately 20:1. Twenty-fourhours later, cells were collected by trypsinization and trans-ferred into six replicate 60-mm culture dishes. Three disheswere fed with medium containing G418 (1 mg/ml), and threewere fed with G418-medium supplemented with emetine-HCI (10-6 M). After 14 days, the culture dishes were fixed in50% (vol/vol) methanol and stained with methylene blue.Biologically active mutant S14 DNAs yielded five- to sixfoldmore colonies in G418-medium than in medium containingG418 plus emetine. In contrast, inactive S14 DNAs yieldedapproximately the same number of colonies (±10%) in thetwo selective media (Fig. 2).

Characterization of mutant S14 transcripts and polypep-tides. To verify that transfected human S14 DNAs wereexpressed efficiently in CHO Emr-2-2 cells, mRNAs andr-proteins were purified from transformed cells after growthin G418 medium. Cytoplasmic S14 mRNAs (14) were as-

sayed by S1 nuclease protection, using an antisense humanRPS14 RNA probe labeled with [32P]UTP to a specificactivity of =5 x 107 cpm/,ug (6, 23). Ribosomal proteinspurified from transfected cells (2) were analyzed by two-dimensional polyacrylamide gel electrophoresis (14, 23).First-dimension tube gels contained 4% (wt/vol) polyacryl-amide, 0.2 M Tris-borate (pH 8.6), 8 M urea, and 10 mMEDTA and were electrophoresed for 2,000 V-h toward thecathode. Second-dimension slab gels included 12.5% (wt/vol) polyacrylamide, 0.1 M BisTris-acetate, 6 M urea, and1% (wt/vol) sodium dodecyl sulfate (pH 6.75) and were

0E

Pir -~

L

VOL. 12, 1992

1682 DIAZ AND ROUFA

A s o n3, B gs fl2MM

.1 *~~~~~~~~1

nss D nv320

FIG. 2. Transfection assay for human RPS14 biological activityCHO Emr-2-2 cells were cotransfected with pSV2Neo and either aDNA encoding a functional RPS14 mutant allele (pMS14-19) (A andC) or an allele containing a missense mutation which inactivates S14(pMS14-31) (B and D). Twenty-four hours later, the cells werereplated into six replicate cultures. Three of the cultures weretreated with G418 (1 mg/ml) (A and B), and three were treated withG418 plus 10' M emetine (C and D). The numbers of colonieswhich developed on each dish after 14 days in culture (n) areindicated.

Exon 11 Exon III ExonIV Exon V

C- TG-. AT-.CA-. G

C-'GG-.TT-AA-.TA- CC-.A'ACC I I T lI1 25 50 75 100 125 150

NudeoddesFIG. 3. Distribution of base substitution mutations recovered in

the human RPS14 protein-coding sequence. The horizontal axisrepresents the S14-coding sequence (453 nucleotides). Transitionmutations are mapped above the axis with lines whose lengthsindicate specific base substitutions (labeled at the left). Transversionmutations are mapped below the axis in a similar manner. S14 exonboundaries are indicated by brackets.

teins in cultured CHO cells. For example, the pattern of S14proteins elaborated by Emr-2-2 cells transfected with mutantDNA pMS14-19 is illustrated in Fig. 4B. It clearly indicatestwo electrophoretically distinguishable S14 polypeptides:the endogenous Emr-2-2 S14 variant (S14a) and a novelr-protein (Sl4b) whose electrophoretic migration was con-sistent with the Arg-55---His mutation encoded by pMS14-19DNA (Table 2). In contrast, transfection of several biologi-cally inactive mutant DNAs did not result in variant S14proteins that could be detected by electrophoresis, despitethe fact that all of the mutated DNAs were transcribedefficiently in CHO cells (see above). pMS14-9 is an examplein which the S14 Cys-31 residue was replaced by Trp (Table

electrophoresed toward the anode for 1,050 V-h. Followingelectrophoresis, gels were fixed in methanol-acetic acid(50%:7.5%) and stained with Coomassie brilliant blue.

RESULTS

In vitro mutagenesis of the human S14-coding sequence.The PCR-mediated chemical mutagenesis strategy describedin Materials and Methods was designed to saturate the entirehuman S14-coding sequence with single-base substitutionmutations. In this way, we isolated 83 DNA clones carrying158 point mutations. As illustrated in Fig. 3, 141 of themutations (89%) were transitions, and 17 (11%) were trans-versions. Mutant S14 DNAs were tested for biologicalactivity by transfection into the emetine-resistant CHO cellline Emr-2-2. Forty-four of the mutant alleles (52%) encodedbiologically active S14 proteins, as they restored Emr-2-2cells to an emetine-sensitive phenotype. The remaining 39DNAs (46%) were judged to encode RPS14 null alleles,because they did not complement drug resistance in trans-formed cells. Efficient transcription of all RPS14 null alleleswas verified by S1 nuclease analysis of transfected cellmessages (data not shown).As illustrated in Fig. 4, the Emr-2-2 S14 protein (S14a) can

be resolved from wild-type S14 (S14c) by two-dimensionalpolyacrylamide gel electrophoresis (Fig. 4A) (13). On thebasis of their altered protein-coding sequences, severalmutant S14 DNAs were predicted to encode new electro-phoretic variants of the S14 polypeptide. This provided abiochemical means to determine whether at least some of themutant DNAs actually were expressed as functional r-pro-

A. W.T.

C.pMS14-9

B.pMS14-19

D.

55 (=< S14bI

517#F-S- 1* ? S14a 14C

320 1: 05

FIG. 4. Polyacrylamide gel electrophoresis of mutant S14 pro-teins expressed in cultured rodent cells. Ribosomal proteins purifiedfrom CHO Emr-2-2 cells stably transformed with wild-type humanS14 DNA (A), with a DNA encoding a functional mutant S14 protein(pMS14-19; B), or with a DNA which specifies a nonfunctionalr-protein (pMS14-9; C) were electrophoresed in two dimensions.Each gel contained the proteins extracted from 5 A260 of ribosomes.As illustrated, the first-dimension electrophoresis was from left toright and the second-dimension was from top to bottom. (A to C) Gelregions which surround r-protein S14. The CHO Emr-2-2 S14polypeptide (13) is labeled S14a, wild-type human S14 is labeledS14c, and the mutant S14 protein encoded by pMS14-19 DNA islabeled S14b. (D) Labeled diagram of the CHO cell r-protein spots.Reference lines connecting r-proteins Lll, L25, and S15 are drawnin each panel.

MOL. CELL. BIOL.

TABLE 2. Point mutations recognized in the human RPS14 protein-coding sequence

Mutation Func- Mutation Func-C

Mutation Func-Clone' tionb loNe tion Clone tionNucleic acid Protein' j Nucleic acid Protein tinNucleic acid Protein to

1 A-76- GT-78 CJA-97 G

2 A-76 GT-179 - C

3 T-91-*A4 C-209-- T5 A-148> G6 C-134 T7 G-63 A

G-92 A8 T-87 - C

T-114 CT-158 CG-168 AT-185 C

9 C-93- G11 T-160-- C

A-182 T12 G-85- A

T-122 - CG-164 A

13 T-109 CA-116 G0

14 C-191 - T15 A-182 G17 C-127 T18 G-85 -*A19 G-164 A20 T-123 C

G-176 - T22 T-96- C

A-152 -- G23 T-87-* C

G-146 -- A24 G-71*A

T-125 - CT-129 CT-158 C

25 C-211 T26 G-197 A

T-201 C27 C-ill T28 A-26 G28a G-27 A29 G-17 A

G-39 A30 T-2 - C

G-40 - AG-61 A

31 T-98 CT-123 C

31a A-24 C32 T--22> C

T- -16 - CG-39 A

33 T-21 C34 A-28> G36 C-8- A37 G-115 -* A39 C--27-*T

C-58 - T41 G-259 A

A-268 GC-320 T

N-26 -- D

1-33 -* VN-26 DM-60 TC-31 SS-70 LK-50 - ET-45 IV-21C-31 YG-29N-38I-53 -* TV-56V-62 AC-31 WC-54 RK-61 - MG-29 SF-41 SR-55 HF-37 LD-39 GA-64 VK-61 - RH-43 YG-29 - SR-55 - HF-41 LG-59 VH-32E-51 G0G-29G-49 DG-24 EV-42 AH-43I-53 TP-71 - SR-66 QD-67F-37K-9 RK-9G-6 EQ-13M-1 - ncV-14 ncV-21 M1-33 -- TF-41E-8ncncQ-13ncdK-10 -- EP-3 - HD-39 NncQ-20 - TrmeE-87 - KI-90 - VT-107 -* I

+

++

++

+

++

++++

+

++

++++

43 C-218 T A-73 - VG-225 A M-75 - IG-243 - A V-81

44 G-258 A K-86G-331 A G-111 R

46 A-310 G R-104 G47 G-229 A A-77 T

G-251-A R-84 -KG-312 A R-104

48 A-248 T Q-83 -* L48a C-289 - T H-9450 C-270 T 1-90

A-300 G T-100C-314 - T T-105 --+I

51 G-232 A A-78 - T52 C-234 T A-78

C-235 T Q-79 TrmA-287 G ncC-289 T nc

53 C-255 T C-85G-293 T R-98 L

54 C-221 T A-74 V55 G-350 A R-117 K56 C-365 T S-122-- L57 C-341- T S-114*F

G-352 A A-118AC-382f -1 FS'

58 G-350 -A R-117-iKC-354 - A A-118G-412 A D-138 N

59 C-365 T S-122 - LG-388 -A E-130 K

60 C-354 T A-118C-360 T A-120G-367 A G-123 S

61 G-358 A A-120 TG-366 A S-122G-367 A G-123 - ST-371 C M-124 TG-383 A R-128 QG-388 A E-130>KT-464 - C nc

62 C-365--T S-122 LG-388 A E-130>K

63 G-366 A S-122G-391 - A D-131 N

64 G-358 A A-120>T65 G-443 A G-148 D66 C-354 - T A-118

C-355 T L-119 FA-360 T A-120C-408 - T P-136C-448 T R-150 C

66a C-363 - T R-12167 G-379 A G-127 R68 G-362 A R-121 H

G-367 A G-123 ST-435 C G-145G-453 A L-151

69 A-24 -) G E-872 G-232 A A-78 -* T73 C-276 - A A-92

A-327 - T G-10974 G-259 A E-87 -> K

C-292 A R-98

+

+++

+

+

+

+

+++

+

75 G-259 A E-87-*K76 C-355 T L-119 F77 AC-382 -1 FS

G-431 A78 G-432 A G-14479 G-354 - T A-11880 T-371 A M-124 K81 C-359 T A-120 V

C-458 T ncC-468 T nc

82 C-360 T A-12083 C-447 T R-14984 G-381 A G-127

G-429 - A K-143G-430-A G-144 R

86 G-295 - T A-99 -+ S88 G-375 - A K-125

T-377 A I-126 -> N89 G-379 A G-127 R

AC-407 -2FSAC-408 JT-467 - A nc

90 C-398--T T-133 IC-414 - T D-138

92 C-355 T L-119--F93 C-360 T A-120

+

++

+

+++

+

+

a Cloned mutant genes (pMS14-1 to pMS14-93).b Mutant alleles' biological activities were assessed by transfection into emetine-resistant CHO EMr-2-2 cells. +, expression of the mutant gene rendered

Emr-2-2 cells sensitive to emetine; -, it did not.Amino acids are specified by their standard single-letter abbreviations. Base substitutions which do not alter the affected codon's amino acid specification (i.e.,

silent mutations) are indicated without an arrow (e.g., V-21).d nc, the mutation affected a noncoding region of the DNA.e Trm, a premature polypeptide termination codon (TAG, TGA, or TAA).f A, single-base deletion.g FS, a frameshift in the protein-coding sequence.

1683

Lil A

1684 DIAZ AND ROUFA

2). It was expected to encode an r-protein with electropho-retic properties similar to those of wild-type S14 (S14c; Fig.4). However, ribosomes purified from cells transfected withpMS14-9 contained only the Emr-2-2 S14 polypeptide (Fig.4C). Thus, substitution of Trp for Cys-31 appears to precludethe mutant S14 protein's assembly into functional 40S ribo-somal subunits.

Table 2 catalogs the structural and functional informationdetermined for all 83 mutant S14 DNAs. Forty-one of theS14 alleles (49%) contained single-base substitution muta-tions, and 42 (51%) possessed between two and sevenmutations each. Three DNAs in the latter group also carriedone- or two-base deletions (pMS14-57, -77, and -89) whichshifted the translational reading frame of downstream pro-tein-coding sequences, and two alleles (pMS14-39 and -52)harbored premature polypeptide termination (nonsense) mu-tations. As expected, the five frameshift and nonsense allelesall were biologically inactive in the CHO cell transfectionassay.

Mutations summarized in Table 2 offer several insightsinto functional aspects of human r-protein S14's primarysequence. For example, the Cys-31 residue in S14 wasmutated to Ser, Tyr, and Trp in mutant alleles pMS14-3, -7,and -9, respectively. In addition, naturally occurring insect,fungal, and plant S14 genes specify Ala at residue 31 (6).Although pMS14-3, -7, and -9 were biologically inactive inthe rodent cell transfection assay (Table 2), the Drosophilamelanogaster RPS14 gene containing Ala-31 previously wasshown to function normally in CHO cells (15). Thus, ourgenetic data indicate that Cys and Ala both satisfy S14'sfunctional requirements at position 31, whereas Ser, Tyr,and Trp do not.

Finally, the distribution of amino acid substitutions withinmutagenized RPS14 alleles suggested that the S14 polypep-tide is organized into several functional domains (labeled Ato E in Fig. 5). Four independent point mutations whichmapped to the first 20 S14 amino acid residues (domain A)did not affect S14 function, demonstrating that the protein'samino terminus can be varied substantially without loss ofbiological activity. In contrast, two protein domains, B andD, appeared to be critical for normal r-protein activity, sincemost single amino acid replacements affecting these regionsresulted in RPS14 null alleles. Domain B resides betweenGly-24 and Gly-49; domain D resides between Thr-107 andAsp-131. Both of the missense mutations in domain B whichare compatible with S14 function carry conservative aminoacid replacements. On the other hand, four of the fivemutations in domain D that do not appear to affect S14activity are associated with nonconservative amino acidsubstitutions. Mutations in domain C (Ile-53 to Thr-105) forthe most part yielded biologically active S14 alleles. Two ofthe four inactive S14 alleles that harbor single point muta-tions within domain C encoded nonconservative amino acidsubstitutions (pMS14-4 and -53), and one carried a prema-ture polypeptide termination mutation (pMS14-52). DomainE (Thr-133 to Leu-151) contains only three point mutations(pMS14-65, -84, and -90) but is the polypeptide regionaffected by amino acid replacements which render CHO cell40S ribosomal subunits resistant to emetine (6, 22). It isnoteworthy that boundaries of the five functional domainsdefined by the distribution of null point mutations corre-spond closely with the S14 gene's protein-coding exons(exons II to V; Fig. 5). Domains A and B are containedwithin exon II, domain C is in exon III, domain D is in exonIV, and domain E is in exon V.The amino terminus of human S14 (domain A) is not

required for the protein's biological activity. pMS14-30 (Table2) is a particularly interesting mutant allele. It includes threepoint mutations which dramatically affect the S14 amino-terminal sequence (Fig. 6). One of the mutations alters thetranslational initiator ATG to ACG and therefore shouldpreclude the normal initiation of protein biosynthesis. Asecond mutation converts the Val-21 codon (GTG) to amethionine codeword (ATG) which, because it resides in afavorable nucleic acid context (11), was expected to restorethe mutant mRNA's activity. The third mutation convertsVal-14 (GTC) to Ile (ATC) upstream of the relocated initiatorcodon. If the pMS14-30 coding sequence were transcribedand translated into a truncated r-protein as proposed in Fig.6, a variant S14 protein initiating at codon 21 might accountfor the DNA's biological activity (Table 2).To test this explanation for the pMS14-30 allele's biolog-

ical activity in transfected CHO cells, we analyzed r-proteinspurified from Emr-2-2 cells stably expressing pMS14-30DNA by two-dimensional electrophoresis (Fig. 7). As shownin Fig. 7A, the pattern of Coomassie blue-stained r-proteinsobserved included an unusual polypeptide (AS14) signifi-cantly smaller and less basic than endogenous Emr-2-2 S14(S14a). In vitro transcription and translation were used todetermine whether AS14 is the protein encoded by pMS14-30. pMS14-30 DNA was excised from pUC13 as a HpaI-HindIII fragment (Fig. 1), subcloned between the SmaI-HindIII sites of pGEM-1, and transcribed by using T7 RNApolymerase. The resulting mRNA was translated in a wheatgerm cell extract containing [35S]methionine (3, 15) to pro-duce a single [35S]polypeptide which comigrated in bothelectrophoretic dimensions with the AS14 protein (Fig. 7B).Therefore, we concluded that AS14 is the truncated polypep-tide encoded by pMS14-30 and that it is assembled intobiologically active, emetine-sensitive Chinese hamster cellribosomes.

Genedc DomainsA B C D E

A

-.. ................................... .. -. ......... 7.. .. .. ....-,,,E- :i-- iif

E ::..-FIG .5 . a.c:::...:eplacementmut.ations distq~~~~~~~~~~~~~~~~~~~~~~~ ..'':.'.''":,'throughout the human SB4 polypEptide.: . .......Th horizo x.....r............

]l.........................................,,,,,., - '.. ....................

FIG.5. Mapof amino acd replacemetmut......di..ribute

sents the 151-amino-acid human S14 polypeptide sequence, with theamino terminus (N) at the left and the carboxyl terminus (C) at theright. Amino acid substitutions which abrogated the r-protein'sbiological activity are indicated above the horizontal axis; mutationswhich were compatible with S14 function are mapped below theaxis. Only mutant alleles that carried single amino acid substitutionsare illustrated. Each mutation is identified by its clone number (seeTable 2). Amino acid substitutions were judged to be conservative(circles) or nonconservative (squares) according to the PAM-250matrix (5). Polypeptide chain termination (nonsense) mutations arerepresented as triangles. S14 structural domains suggested by thedistribution of missense mutations are shaded and labeled A to B.RPS14 protein-coding exons II to V are represented as rectanglesbelow the map.

MOL. CELL. BIOL.

FINE-STRUCTURE MAP OF RPS14 1685

A PromoterI

11 - VI IATG ACG ||GTG/ o ATG,~~~~~~~~~l

TranB

scription Translation

1 2 3 4 5 6 7 8 9 10 11 12 13

Met Ala Pro Arg Lys Gly Lys Glu Lys Lys Glu Glu Glnwild Type ATG GCA CCT CGA AAG GGG AAG GAA AAG AAG GAA GAA CAG

pIUL4 -30 AQG GCA CCT CGA AAG GGG AAG GAA AAG AAG GAA GAA CAG

14 15 16 17 18 19 20 21 22 23 24 25 26

Val Ile Ser Leu Gly Pro Gln Val Ala Glu Gly Glu AsnWild Type GTC ATC AGC CTC GGA CCT CAG GTG GCT GAA GGA GAG AAT

I I

pI14-30 ATC ATC AGC CTC GGA CCT CAG ATG GCT GAA OGA GAG AAT ...

Hat Ala Glu Gly Glu Asn ...

FIG. 6. (A) Diagram showing that mutant allele pMS14-30 en-codes a truncated r-protein which lacks the amino terminus ofwild-type S14 protein. The mutant S14 sequence encoded bypMS14-30 is depicted. Rectangles represent the gene's exons (la-beled in roman); lines represent the upstream flanking and intronicDNA sequences. The original initiator ATG codon, located 3 bpfrom the 5' end of exon II, was mutated to an ACG codeword.Simultaneously, the codon for Val-21 (GTG) was mutated to areplacement initiator codeword (ATG). As a result of both muta-tions, the pMS14-30 polypeptide was predicted to lack the first 20amino acid residues contained in wild-type S14 (hatched box). (B)DNA and amino acid sequences involved in these mutations.

DISCUSSION

PCR-mediated nitrous acid mutagenesis (7) was used tomutate the human r-protein S14-coding sequence. Eighty-three RPS14 alleles were isolated as recombinant DNAclones. We determined nucleotide sequence alterations car-ried by each mutated DNA as well as its encoded r-protein'sbiological activity after transfection into cultured rodentcells. In all, we recognized 158 base substitution mutations.Slightly more than half of the mutant alleles (52%) werebiologically active, as they restored emetine-resistant CHOcells to the wild-type (i.e., drug-sensitive) phenotype. Theremaining alleles (48%) encoded r-proteins which phenotyp-ically were inactive in the CHO cell transfection assay.

Nucleotide sequence data were used to identify and mapthe amino acid substitutions encoded by mutagenizedDNAs. The mutations characterized resolved five distinctS14 polypeptide domains (labeled A to E in Fig. 5), two ofwhich (domains B and D) appeared to be important forr-protein biological activity. Remarkably, the polypeptidestructural domains corresponded quite closely with theboundaries of human RPS14 exons II to V. Others haveproposed that modern eukaryotic exons may have derivedfrom a limited number of ancestral exons whose recruitmentinto diverse loci provided an efficient mechanism for proteinevolution (8-10, 16, 20). The correlation between humanRPS14's mutationally defined functional domains and itsexon structure is consistent with this hypothesis.

Several yeast r-proteins contain nuclear localization sig-nals (NLSs) close to their amino termini (1, 17, 28). Theyeast r-protein NLSs are short, basic peptide motifs thatdisplay striking similarity to the simian virus 40 (SV40)T-antigen NLS, PKKKRKV (12). More recently, an NLShas been recognized within an internal domain of rat r-pro-tein S2; although it is also a short basic peptide motif(RGGF), the S2 NLS does not resemble the SV40 T-antigen

A. B.

FIG. 7. Assembly of the r-protein encoded by pMS14-30 intofunctional CHO cell ribosomes. The figure illustrates a two-dimen-sional polyacrylamide gel electrophoresis of r-proteins purified fromCHO Emr-2-2 cells transfected with pMS14-30 DNA. (A) Coomassieblue-stained pattern of proteins observed in the S14 region of thegel; (B) diagram identifying each spot and an overlay of theautoradiogram used to detect the pMS14-30 polypeptide synthesizedin vitro. The r-protein labeled AS14 below and to the right of the S20protein (A) was not observed in ribosomes purified from controlCHO Emr-2-2 cells (Fig. 4). However, this protein comigrates withthe radioactive cell-free polypeptide product encoded by pMS14-30DNA (B).

NLS (27). Mammalian S14 lacks peptide motifs with obvioussimilarity to either the SV40 T-antigen or human r-protein S2NLS sequences, despite the fact the S14 protein is a verybasic polypeptide (Arg + Lys = 18%) (23). In addition,because the amino-terminal 20 residues of human S14 (do-main A) are functionally dispensable, domain A is unlikely toencode a necessary NLS. On the other hand, most of themissense mutations mapped to domains B (Gly-24 to Gly-49)and D (Thr-107 to Asp-131) abrogated S14's biological activ-ity, indicating that these regions of the r-protein are criticalfor its normal function. Experiments to determine whetherS14 domains B and/or D might encode an NLS now are inprogress.

In a previous attempt to isolate null alleles of -the mam-malian RPS14 gene by tissue culture mutagenesis experi-ments, we surveyed large numbers of ethyl methanesulfo-nate-treated CHO cells carrying extra copies of a clonedemetine resistance RPS14 allele. The somatic genetic ap-proach yielded only six point mutations that destroyed S14'sbiological activity (6). Four of the null alleles isolated in vivoencoded single amino acid replacements (Emr-36, Gly-111--Arg; Emr-39, Gly-127-->Arg; Emr-75, Arg-150-+Gly;and Emr-80, Cys-85--*Phe), and two carried nonsense muta-tions (Emr-77, Gln-20 [CAA]--TAA; and Emr-84, Arg-98[CGA]-*TGA). The low recovery of null alleles from tissueculture selection experiments indicated either that most ofS14's amino acid residues might be mutated without loss ofbiological activity or that the majority of S14 missensemutations might confer a dominant lethal phenotype whichcould not be isolated in living cells. Data described in thisreport demonstrate that (i) 47 amino acid substitutionsisolated in vitro abrogated S14's activity without conferringa dominant lethal phenotype on cultured rodent cells and (ii)at least 32 different amino acid substitutions were compatiblewith a sufficient level of S14 function to complement theEmr-2-2 emetine resistance mutation (Table 2).

ACKNOWLEDGMENTS

We express our appreciation to Dawn Slifer for expert help withtissue culture and to Carl G. Maki, Beth A. Montelone, and DavidA. Rintoul for help during preparation of the manuscript.

J.J.D. is a Postdoctoral Cancer Research Fellow of The Wesley

VOL. 12, 1992

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1686 DIAZ AND ROUFA

Foundation of Wichita, Kans. Support for this study was providedby NIH grants GM38932 and GM23013.

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