Gene, 107 (1931) 329-333 0
1991 Elsevier
GENE
Science
Publishers
B.V. All rights reserved.
329
0378-l 119/91/$03.50
06086
Cloning and sequencing a cDNA encoding -human ribosomal protein S25 (Recombinant
DNA;
Maolin Li, Chantal
subtractive
Latoud
cDNA
library;
synthetic
peptide;
conserved
sequence)
and Melvin S. Center
Center for Basic Cancer Researdz, L&&ion of Biology, Kmms
State W~~ve~~~~,~~~~atta~,
KS 66.506 (U.S.A.)
Received by J.A. Engler: 24 May 1991 Accepted: 29 July 1991 Received at publishers: 9 August 199 1
A full-length cDNA clone has been isolated from a cDNA library prepared from mRNA of adriamycin-resistant human leukemia HL60 cells. The nucleotide sequence of this cDNA has been determined and the protein coded for by the gene identified. The cDNA encodes a polypeptide of 125 amino acids (aa) with a deduced M, of 13 750. The deduced aa sequence of this protein has 56% homology to yeast ribosomal protein S31. Western-blot analysis using antibodies directed against a synthetic peptide based on the deduced aa sequence identities the gene product as the human ribosomal protein S25.
INTRODUCTION
RESULTS
Eukaryotic ribosomes consist of four rRNAs and 80 r-proteins in the mature 40s and 60s subunits. Synthesis of an equal amount of r-proteins and rRNAs is coordinately controlled (Mager, 1988). Analysis of the genes which code for r-proteins may lead to an und~rst~ding of the function and structure of ribosomes. The coding sequences of a number of r-protein genes have been isolated and characterized (Nomura et al., 1984; Leer et al., 1984; Rhoads et al., 1986; Suzuki et al., 1990). The aim of the present study was the cloning and nucleotide sequence determination of a eDNA encoding human r-protein S25.
(a) Sequence
Correspondence
z‘o: Dr. M.S. Center,
Division
of Biology,
Kansas
State
University, Manhattan, KS 66506 (U.S.A.) Tel. (913)532-6&l; Fax (913)5X2-6653. Abbreviations:
aa, amino acid(s); bp, base pair(s);
mentary to mRNA; 2-D, two-dimensional; reading frame; PAGE, polyacrylamide-gel SDS, sodium
dodecyl
sulfate.
cDNA, DNA comple-
nt, nucleotide(s); ORF, open electrophoresis; r, ribosomal;
AND DISCUSSION
of human
r-protein
S25
A subtractive cDNA library (Tedder et al., 1988) was constructed in phage dgtl0 starting with mRNA of HL.60 drug-sensitive and -resistant cells. Genes which are overexpressed in drug-resistant cells would be expected to be enriched in this cDNA library. mRNA isolated from both cell lines was used to synthesize a radioactively labeled single-stranded cDNA. This material was used as a probe to screen the cDNA library. Analysis of several cDNA clones demonstrated that one contained sequences which are overexpressed in drug-resistant cells. These data will be published elsewhere. This cDNA clone (PCR8) was isolated and characterized. The cDNA was subcloned into plasmid pGEM- 11Zf and the nt sequence was determined using the dideoxy chain-termination method (Sanger et al., 1977). The nt sequence and deduced aa sequence are shown in Fig. 1. The ORF encodes a polypeptide of 125 aa that begins at an AI-G codon. The calculated II& of the deduced protein is 13750. The 3’-untranslated region of PC-R8 cDNA contains a 58-bp nontranslated region with a polyadenylation signal of AATAAAA and ATTAAA at nt positions 408-413 and 419-424 and a poly(A) tail. The S/-untranslated region of PC.R8 cDNA has a poly(T) tract.
330 -61 TTTTTTTTTTT -1
-30 TTTTTGTCCGACATCTTGAGACGAGGCTGCGGTGTCTGCTTT~~~CGAGCTTCG~A
1 30 ATGCCGCCTAAGGACGACAAGAAGAAGAAGGACGCTGGAAAGTCGGCCAAGAAAGACAAA MetProProLysAspAspLysLysLysLysAspAlaGlyLysSerAl~LysLysAspLys
60
90 GACCCAGTGAACAAATCCGGGGGCAAGGCCAAAAAGAAGAAGTGGTCCAAAGGCAAAGTT AspProValAsnLysSerGlyGlyLysAlaLysLysLysLysTrpSerLysGlyLysVal
120
150 CGGGACAAGCTCAATAACTTAGTCTTGTTTGACAAAGCTACTCTGTAAG ArgAspLysLeuAsnAsnLeuValLeuPheAspLysAlaThrTyrAspLysL~uCysLys
180
210
240
GAAGTTCCeAACTATAAACTTATMCCCCAGCTGTGGTCTCTGAGAGACTG~GATTCGA GluVafProAsnTyrLysLeuIleThrProAlaValValSerGluArgLeuLysIl~Arg 270 GGCTCCCTGGCCAGGGCAGCCCTTCAGTACTGGTT GlySerLeuAlaArgAlaAlaLeuGlnGlnGlu~euLeuS~~LysGlyLe~~leLysLeuVal
300
330 TCAAAGCACAGAGCTCAAGTAATTTTA~ACCPGGGTGGAGATGCTCCAGCT SerLysHisArgalaGlnValIleTyrThrArgAsnThrLysGlyG~yAspAlaProAla
360
390 GCTGGTGAAGATGCATG~TAGGTCCAACCACCAGCTGTACATTTGG~TA~CTTT~ AlaGlyGluAspAla***
420
TAAATC-. Fig. 1. The nt sequence
. .
of cloned cDNA and the deduced
-resistant
human leukemia HMO mRNA. A subtractive
screening
of the cDNA
library. The nt sequence
aa sequence
ofthe
encoded
cDNA library was constructed
of this cDNA clone was determined
protein.
cDNAs
were synthesized
from adr~~ycin-sensitive
(Tedder et al., 1988) and acDNA using the dideoxy
and
clone was isolated by subtractive
chain-termination
method
(Sanger
et al., 1977).
The nt residues are numbered from the 5’ to 3’ end and aa are numbered starting with position 1 in the ORF. Last digits of numerals are aligned with corresponding nt. The stop codon is designated with three asterisks. Polyadenylation processing signals are underlined. This nt sequence has been deposited
with GenBank
Human
under accession
S-25
No. M64716.
MPPKDDKKKKDAGKSAKKDKDPVNKSGGKAK-KKKWSKGK ..* ..r.. ** **. .*...I * *** * ...*m.* s.. ------GGK-KSKKKWSK-K MPPKQQLS~-A-~~
Yeast
S-31
Human
S-25
Yeast
S-31
Human
S-25
Yeast
S-31
Human
S-25
Yeast
S-31
. a
l
l
-VRDKLNNLV-LFD-.. .* .. *. SMKD~Q~AVIL-DQEK
KATYDK-LCKEVPNYKLITPAV-V .. 1). .. .m** : ** .** --YDRIL-KEVPTYRYVSVSVLV l
SERLKIRGSL,ARAALQELLSK-GLIKLVSKHRAQVIYTRN .*.. ** ** .e .. .. a*. *.** .*.... . . . . .* .a I.. -~RLKIGGS~RIALRHLE-KEG~~KPISKHSKQAIYT~ TKGGDAPAAGEDA : : .. T----AS---E--
39 30
..
..
73 66
112 :
: : : : 104 125 108
Fig. 2. Comparison of human r-protein 525 and yeast r-protein 531. The aa sequence of human r-protein deduced from full-length PCRX cDNA (see Fig. 1) is aligned with the aa sequence of yeast r-protein S31 deduced from a genomic DNA clone (Nieuwint et al., 1985). Gaps (dashes) are introduced to maximize
sequence
alignment.
Identical
aa are indicated
by colons.
PAGE and Western-blot analysis of hnman r-proteins. The 2-R PAGE of human r-proteins was carried out acco rding to the Fig. 3. Two-dimensional tube gel (left to right) consisted of 4% (w/v) polyacrylamide in 0.2 M Tris 1borai tef8 M proc :edure described by Madjar et al. (1983). The first-dimension slab gel (top to bottom) contained 12.5% polyac~lam~de in 0.1 h4 bis Tris acetate pH 6.75/6 M urea, 10.2% (w/v) ure.aLpH 8.6. The second-dimension
332 Recent studies suggest that 5’-polypyrimidine regions in mRNA may be involved in translational control of r-protein synthesis (Levy et al., 1991).
this peptide was used as a probe to identify the cDNA gene product.
(b) Sequence homology with yeast r-protein S31
Human r-proteins were isolated according to the procedure described by Nakamichi et al. (1983) and thereafter analyzed by two-dimensional polyacrylamide gel electro-
A search of GenBank (Bilofsky and Burks, 1988) data sequence similarities with PC&R8 cDNA shows a major sequence homology with a previously isolated yeast genomic r-protein S31 DNA (Nieuwint et al., 1985). Align-
phoresis (Madjar et al., 1983). The 2-D gel pattern of Coomassie blue-stained r-proteins and the result of a Western-blot analysis using the antipeptide serum are shown in Fig. 3. The au~oradiographic spot detected in the
ment of the deduced
Western blot (Fig. 3b) matches well with a 525 protein spot detected in the Coomassie blue-stained gel (Fig. 3a). The proteins of the Coomassie blue-stained gel are numbered according to the standard eukaryotic r-protein nomencla-
aa sequences
of yeast S3 1 and PC.RS
is shown in Fig. 2. The two polypeptides have 61 aa identities in 108 possible matches (56%). This result suggests that these two proteins may have a common ancestor.
ture (McConkey (c) Identification of human r-protein 525 A peptide of 15 aa contained in the C terminus of the deduced aa sequence of PC.RS cDNA was synthesized by the Merrified solid-state method (Marglin and Merrifield, 1970). The synthetic peptide cont~ning Lys at the N terminus was conjugated to keyhole limpet hemocyanin and used to produce antiserum in a rabbit. Antiserum against
TABLE
I
Comparison
of aa compositions
of human
et al., 1979; Brown
et al., 1990).
In previous studies Lin et al. (1979) determined the aa composition of the rat r-protein 525. A comparison of the aa compositions of rat S2.5 with that determined from the deduced sequence of PCRS is given in Table I. It is clear that the aa composition is essentially the same for the two proteins. These results, taken together with the 2-D Western-blot analysis, strongly suggest that the PC-R8 cDNA isolated in the present study codes for the human r-protein S25.
525 and rat S2S ACKNOWLEDGEMENTS Rat S25b
aa
Human
S25”
Ala
10.4
10.5
Cys Asp Gill
0.8 8.0
ND
3.2
12.7 6.2
Phe
We are grateful to Dr. Donald J. Roufa for advice and helpful discussion and to Carl Maki for his technical assistance. This investigation was supported by Research Grant CA37585 from the National Cancer Institute, Department of Health and Human services.
Gly His Ile
0.8 1.2
1.0 10.7
0.8
0.8 2.9
LYs Leu
21.6 9.6
20.3
Met Asn
0.8 4.0
0.0 NR
Bilofsky, H.S. and Burks, C.: The GenBank genetic sequence Nucleic Acids Res. 16 (1988) 1861-1864.
Pro Gln
4.8 1.6
3.2 NR
Brown, S.J., Jewel& A., Maki, C.G. and Roufa, D.J.: A cDNA encoding human ribosomal protein S24. Gene 91 (1990) 293-296.
Arg Ser
4.8 5.6
Thr Val
5.0 6.3 3.2
6.4
Trp Tyr
0.8 2.4
3.2
3.2
“The aa composition b The aa composition ribosomal
protein
REFERENCES
8.8
Leer,R.J., W.H.
genes. Nucleic
Acids Res. 12 (1984) 6685-6700.
Levy, S., Avni, D., Hariharan,
N., Perry, R.P. and Meyuhas,
0.: Oligo-
pyrimidine tract at the 5’ end of mammalian ribosomal protein mRNAs is required for their translational control. Proc. Natl. Acad. Sci. USA 88 (1991) 3319-3323.
1.8
(mol%> determined from the present study. (mol%) as described by Lin et al. (1979) for rat
525. ND, not determined;
Van Kaamsdonk-Duin, M.M.C., Hagendoorn, M.J.M., Mager, and Pianta, R-J.: Structure ~ornp~iso~ of yeast ribosomal
protein
6.6 ND
data bank.
Lin, A., Tanaka, T. and Wool, LG.: Isolation proteins: p~ifi~ation and characterization chemistry
NR, not reported.
of eukaryotic ribosomal of 325 and L16. Bio-
18 (1979) 1634-1637.
-. SDS. Panel A shows the results of a 2-D gel of r-proteins stained with Coomassie blue. Panel B shows the results of a Western-blot analysis in which proteins of the 2-D gel were transferred to nitrocellulose as described by Towbin et al. (1977). After blocking with 2% bovine serum albumin, the filter was incubated for 12 h with antiserum against a C-terminal synthetic peptide prepared according to the deduced sequence of the protein encoded by PCRX. The filters were thereafter washed and incubated with Lz51-labeled protein A (5 x 10’ cpmjml) for 2 h. The filters were extensively washed and after drying immunoreactive
protein
was detected
by autoradiography.
Arrows
indicate
the position
to which human
r-protein
525 migrates.
333 Madjar,
J.-J., Frahm,
M., McGill,
S.M. and Roufa,
protein S 14 is altered by two-step emetine resistance hamster cells. Mol. Cell. Biol. 3 (1983) 190-197. Mager, W.H.: Control of ribosomal Biophys. Acta 949 (1988) 1-15. Marglin,
A. and Merritield,
protein
R.B.: Chemical
D.J.: Ribosomal genes in Chinese
gene expression. synthesis
Biochem.
of peptides
and
proteins. Annu. Rev. Biochem. 39 (1970) 841-866. McConkey, E.H., Bielka, H., Gordon, J., Lastick, S.M., Lin, A., Ogata, K., Reboud, J.-P., Traugh, J.A., Tram, R.R., Warner, J.R., Welfle, H. and Wool, I.G.: Proposed uniform nomenclature for mammalian ribosomal proteins. Mol. Gen. Genet. 169 (1979) l-6. Nakamichi, N., Rhoads, D.D. and Roufa, D.J.: The Chinese hamster
cell
emetine resistance gene: analysis of cDNA and genomic sequences encoding ribosomal protein S14. J. Biol. Chem. 258 (1983) 13236-13242. Nieuwint, R.T.M.,
Molenaar,
C.M.T.,
Van
Bommel,
J.H.,
Van
Raamsdonk-Duin, M.M.C., Mager, W.H. and Planta, R.J.: The gene for yeast ribosomal protein S31 contains an intron in the leader sequence.
Curr. Genet.
10 (1985) 1-5.
Nomura,
M., Gourse,
R. and Baughman,
of ribosomes and ribosomal (1984) 75-l 17. Rhoads,
G.: Regulation
components.
D.D., Dixit, A. and Roufa,
D.J.: Primary
structure
ribosomal protein S14 and the gene that encodes 6 (1986) 2774-2783. Sanger,
F., Nicklen,
terminating
S. and Coulson,
inhibitors.
5463-5461. Suzuki, K., Olvera,
Proc.
J. and Wool,
of the synthesis
Annu. Rev. Biochem.
Acad.
Sci.
LG.: The primary
of human
it. Mol. Cell. Biol.
A.R.: DNA sequencing Natl.
USA
with chain74 (1977)
structure
of rat
ribosomal protein L9. Gene 93 (1990) 297-300. Tedder, T.F., Strueli, M., Schlossman, S.F. and Saito, H.: Isolation structure of a cDNA encoding the B 1 (CD20) cell-surface human B lymphocytes. Proc. Natl. Acad. Sci. USA 208-211. Towbin, H., Staehelin,
T. and Gordon,
53
J.: Electrophoretic
and
antigen of 85 (1988) transfer
of
proteins from polyacrylamide gels to nitrocellulose sheet: procedure and some applications. Proc. Natl. Acad. Sci. USA 76 (1979) 4350-4354.
1991 Elsevier
GENE
Science
Publishers
B.V. All rights reserved.
329
0378-l 119/91/$03.50
06086
Cloning and sequencing a cDNA encoding -human ribosomal protein S25 (Recombinant
DNA;
Maolin Li, Chantal
subtractive
Latoud
cDNA
library;
synthetic
peptide;
conserved
sequence)
and Melvin S. Center
Center for Basic Cancer Researdz, L&&ion of Biology, Kmms
State W~~ve~~~~,~~~~atta~,
KS 66.506 (U.S.A.)
Received by J.A. Engler: 24 May 1991 Accepted: 29 July 1991 Received at publishers: 9 August 199 1
A full-length cDNA clone has been isolated from a cDNA library prepared from mRNA of adriamycin-resistant human leukemia HL60 cells. The nucleotide sequence of this cDNA has been determined and the protein coded for by the gene identified. The cDNA encodes a polypeptide of 125 amino acids (aa) with a deduced M, of 13 750. The deduced aa sequence of this protein has 56% homology to yeast ribosomal protein S31. Western-blot analysis using antibodies directed against a synthetic peptide based on the deduced aa sequence identities the gene product as the human ribosomal protein S25.
INTRODUCTION
RESULTS
Eukaryotic ribosomes consist of four rRNAs and 80 r-proteins in the mature 40s and 60s subunits. Synthesis of an equal amount of r-proteins and rRNAs is coordinately controlled (Mager, 1988). Analysis of the genes which code for r-proteins may lead to an und~rst~ding of the function and structure of ribosomes. The coding sequences of a number of r-protein genes have been isolated and characterized (Nomura et al., 1984; Leer et al., 1984; Rhoads et al., 1986; Suzuki et al., 1990). The aim of the present study was the cloning and nucleotide sequence determination of a eDNA encoding human r-protein S25.
(a) Sequence
Correspondence
z‘o: Dr. M.S. Center,
Division
of Biology,
Kansas
State
University, Manhattan, KS 66506 (U.S.A.) Tel. (913)532-6&l; Fax (913)5X2-6653. Abbreviations:
aa, amino acid(s); bp, base pair(s);
mentary to mRNA; 2-D, two-dimensional; reading frame; PAGE, polyacrylamide-gel SDS, sodium
dodecyl
sulfate.
cDNA, DNA comple-
nt, nucleotide(s); ORF, open electrophoresis; r, ribosomal;
AND DISCUSSION
of human
r-protein
S25
A subtractive cDNA library (Tedder et al., 1988) was constructed in phage dgtl0 starting with mRNA of HL.60 drug-sensitive and -resistant cells. Genes which are overexpressed in drug-resistant cells would be expected to be enriched in this cDNA library. mRNA isolated from both cell lines was used to synthesize a radioactively labeled single-stranded cDNA. This material was used as a probe to screen the cDNA library. Analysis of several cDNA clones demonstrated that one contained sequences which are overexpressed in drug-resistant cells. These data will be published elsewhere. This cDNA clone (PCR8) was isolated and characterized. The cDNA was subcloned into plasmid pGEM- 11Zf and the nt sequence was determined using the dideoxy chain-termination method (Sanger et al., 1977). The nt sequence and deduced aa sequence are shown in Fig. 1. The ORF encodes a polypeptide of 125 aa that begins at an AI-G codon. The calculated II& of the deduced protein is 13750. The 3’-untranslated region of PC-R8 cDNA contains a 58-bp nontranslated region with a polyadenylation signal of AATAAAA and ATTAAA at nt positions 408-413 and 419-424 and a poly(A) tail. The S/-untranslated region of PC.R8 cDNA has a poly(T) tract.
330 -61 TTTTTTTTTTT -1
-30 TTTTTGTCCGACATCTTGAGACGAGGCTGCGGTGTCTGCTTT~~~CGAGCTTCG~A
1 30 ATGCCGCCTAAGGACGACAAGAAGAAGAAGGACGCTGGAAAGTCGGCCAAGAAAGACAAA MetProProLysAspAspLysLysLysLysAspAlaGlyLysSerAl~LysLysAspLys
60
90 GACCCAGTGAACAAATCCGGGGGCAAGGCCAAAAAGAAGAAGTGGTCCAAAGGCAAAGTT AspProValAsnLysSerGlyGlyLysAlaLysLysLysLysTrpSerLysGlyLysVal
120
150 CGGGACAAGCTCAATAACTTAGTCTTGTTTGACAAAGCTACTCTGTAAG ArgAspLysLeuAsnAsnLeuValLeuPheAspLysAlaThrTyrAspLysL~uCysLys
180
210
240
GAAGTTCCeAACTATAAACTTATMCCCCAGCTGTGGTCTCTGAGAGACTG~GATTCGA GluVafProAsnTyrLysLeuIleThrProAlaValValSerGluArgLeuLysIl~Arg 270 GGCTCCCTGGCCAGGGCAGCCCTTCAGTACTGGTT GlySerLeuAlaArgAlaAlaLeuGlnGlnGlu~euLeuS~~LysGlyLe~~leLysLeuVal
300
330 TCAAAGCACAGAGCTCAAGTAATTTTA~ACCPGGGTGGAGATGCTCCAGCT SerLysHisArgalaGlnValIleTyrThrArgAsnThrLysGlyG~yAspAlaProAla
360
390 GCTGGTGAAGATGCATG~TAGGTCCAACCACCAGCTGTACATTTGG~TA~CTTT~ AlaGlyGluAspAla***
420
TAAATC-. Fig. 1. The nt sequence
. .
of cloned cDNA and the deduced
-resistant
human leukemia HMO mRNA. A subtractive
screening
of the cDNA
library. The nt sequence
aa sequence
ofthe
encoded
cDNA library was constructed
of this cDNA clone was determined
protein.
cDNAs
were synthesized
from adr~~ycin-sensitive
(Tedder et al., 1988) and acDNA using the dideoxy
and
clone was isolated by subtractive
chain-termination
method
(Sanger
et al., 1977).
The nt residues are numbered from the 5’ to 3’ end and aa are numbered starting with position 1 in the ORF. Last digits of numerals are aligned with corresponding nt. The stop codon is designated with three asterisks. Polyadenylation processing signals are underlined. This nt sequence has been deposited
with GenBank
Human
under accession
S-25
No. M64716.
MPPKDDKKKKDAGKSAKKDKDPVNKSGGKAK-KKKWSKGK ..* ..r.. ** **. .*...I * *** * ...*m.* s.. ------GGK-KSKKKWSK-K MPPKQQLS~-A-~~
Yeast
S-31
Human
S-25
Yeast
S-31
Human
S-25
Yeast
S-31
Human
S-25
Yeast
S-31
. a
l
l
-VRDKLNNLV-LFD-.. .* .. *. SMKD~Q~AVIL-DQEK
KATYDK-LCKEVPNYKLITPAV-V .. 1). .. .m** : ** .** --YDRIL-KEVPTYRYVSVSVLV l
SERLKIRGSL,ARAALQELLSK-GLIKLVSKHRAQVIYTRN .*.. ** ** .e .. .. a*. *.** .*.... . . . . .* .a I.. -~RLKIGGS~RIALRHLE-KEG~~KPISKHSKQAIYT~ TKGGDAPAAGEDA : : .. T----AS---E--
39 30
..
..
73 66
112 :
: : : : 104 125 108
Fig. 2. Comparison of human r-protein 525 and yeast r-protein 531. The aa sequence of human r-protein deduced from full-length PCRX cDNA (see Fig. 1) is aligned with the aa sequence of yeast r-protein S31 deduced from a genomic DNA clone (Nieuwint et al., 1985). Gaps (dashes) are introduced to maximize
sequence
alignment.
Identical
aa are indicated
by colons.
PAGE and Western-blot analysis of hnman r-proteins. The 2-R PAGE of human r-proteins was carried out acco rding to the Fig. 3. Two-dimensional tube gel (left to right) consisted of 4% (w/v) polyacrylamide in 0.2 M Tris 1borai tef8 M proc :edure described by Madjar et al. (1983). The first-dimension slab gel (top to bottom) contained 12.5% polyac~lam~de in 0.1 h4 bis Tris acetate pH 6.75/6 M urea, 10.2% (w/v) ure.aLpH 8.6. The second-dimension
332 Recent studies suggest that 5’-polypyrimidine regions in mRNA may be involved in translational control of r-protein synthesis (Levy et al., 1991).
this peptide was used as a probe to identify the cDNA gene product.
(b) Sequence homology with yeast r-protein S31
Human r-proteins were isolated according to the procedure described by Nakamichi et al. (1983) and thereafter analyzed by two-dimensional polyacrylamide gel electro-
A search of GenBank (Bilofsky and Burks, 1988) data sequence similarities with PC&R8 cDNA shows a major sequence homology with a previously isolated yeast genomic r-protein S31 DNA (Nieuwint et al., 1985). Align-
phoresis (Madjar et al., 1983). The 2-D gel pattern of Coomassie blue-stained r-proteins and the result of a Western-blot analysis using the antipeptide serum are shown in Fig. 3. The au~oradiographic spot detected in the
ment of the deduced
Western blot (Fig. 3b) matches well with a 525 protein spot detected in the Coomassie blue-stained gel (Fig. 3a). The proteins of the Coomassie blue-stained gel are numbered according to the standard eukaryotic r-protein nomencla-
aa sequences
of yeast S3 1 and PC.RS
is shown in Fig. 2. The two polypeptides have 61 aa identities in 108 possible matches (56%). This result suggests that these two proteins may have a common ancestor.
ture (McConkey (c) Identification of human r-protein 525 A peptide of 15 aa contained in the C terminus of the deduced aa sequence of PC.RS cDNA was synthesized by the Merrified solid-state method (Marglin and Merrifield, 1970). The synthetic peptide cont~ning Lys at the N terminus was conjugated to keyhole limpet hemocyanin and used to produce antiserum in a rabbit. Antiserum against
TABLE
I
Comparison
of aa compositions
of human
et al., 1979; Brown
et al., 1990).
In previous studies Lin et al. (1979) determined the aa composition of the rat r-protein 525. A comparison of the aa compositions of rat S2.5 with that determined from the deduced sequence of PCRS is given in Table I. It is clear that the aa composition is essentially the same for the two proteins. These results, taken together with the 2-D Western-blot analysis, strongly suggest that the PC-R8 cDNA isolated in the present study codes for the human r-protein S25.
525 and rat S2S ACKNOWLEDGEMENTS Rat S25b
aa
Human
S25”
Ala
10.4
10.5
Cys Asp Gill
0.8 8.0
ND
3.2
12.7 6.2
Phe
We are grateful to Dr. Donald J. Roufa for advice and helpful discussion and to Carl Maki for his technical assistance. This investigation was supported by Research Grant CA37585 from the National Cancer Institute, Department of Health and Human services.
Gly His Ile
0.8 1.2
1.0 10.7
0.8
0.8 2.9
LYs Leu
21.6 9.6
20.3
Met Asn
0.8 4.0
0.0 NR
Bilofsky, H.S. and Burks, C.: The GenBank genetic sequence Nucleic Acids Res. 16 (1988) 1861-1864.
Pro Gln
4.8 1.6
3.2 NR
Brown, S.J., Jewel& A., Maki, C.G. and Roufa, D.J.: A cDNA encoding human ribosomal protein S24. Gene 91 (1990) 293-296.
Arg Ser
4.8 5.6
Thr Val
5.0 6.3 3.2
6.4
Trp Tyr
0.8 2.4
3.2
3.2
“The aa composition b The aa composition ribosomal
protein
REFERENCES
8.8
Leer,R.J., W.H.
genes. Nucleic
Acids Res. 12 (1984) 6685-6700.
Levy, S., Avni, D., Hariharan,
N., Perry, R.P. and Meyuhas,
0.: Oligo-
pyrimidine tract at the 5’ end of mammalian ribosomal protein mRNAs is required for their translational control. Proc. Natl. Acad. Sci. USA 88 (1991) 3319-3323.
1.8
(mol%> determined from the present study. (mol%) as described by Lin et al. (1979) for rat
525. ND, not determined;
Van Kaamsdonk-Duin, M.M.C., Hagendoorn, M.J.M., Mager, and Pianta, R-J.: Structure ~ornp~iso~ of yeast ribosomal
protein
6.6 ND
data bank.
Lin, A., Tanaka, T. and Wool, LG.: Isolation proteins: p~ifi~ation and characterization chemistry
NR, not reported.
of eukaryotic ribosomal of 325 and L16. Bio-
18 (1979) 1634-1637.
-. SDS. Panel A shows the results of a 2-D gel of r-proteins stained with Coomassie blue. Panel B shows the results of a Western-blot analysis in which proteins of the 2-D gel were transferred to nitrocellulose as described by Towbin et al. (1977). After blocking with 2% bovine serum albumin, the filter was incubated for 12 h with antiserum against a C-terminal synthetic peptide prepared according to the deduced sequence of the protein encoded by PCRX. The filters were thereafter washed and incubated with Lz51-labeled protein A (5 x 10’ cpmjml) for 2 h. The filters were extensively washed and after drying immunoreactive
protein
was detected
by autoradiography.
Arrows
indicate
the position
to which human
r-protein
525 migrates.
333 Madjar,
J.-J., Frahm,
M., McGill,
S.M. and Roufa,
protein S 14 is altered by two-step emetine resistance hamster cells. Mol. Cell. Biol. 3 (1983) 190-197. Mager, W.H.: Control of ribosomal Biophys. Acta 949 (1988) 1-15. Marglin,
A. and Merritield,
protein
R.B.: Chemical
D.J.: Ribosomal genes in Chinese
gene expression. synthesis
Biochem.
of peptides
and
proteins. Annu. Rev. Biochem. 39 (1970) 841-866. McConkey, E.H., Bielka, H., Gordon, J., Lastick, S.M., Lin, A., Ogata, K., Reboud, J.-P., Traugh, J.A., Tram, R.R., Warner, J.R., Welfle, H. and Wool, I.G.: Proposed uniform nomenclature for mammalian ribosomal proteins. Mol. Gen. Genet. 169 (1979) l-6. Nakamichi, N., Rhoads, D.D. and Roufa, D.J.: The Chinese hamster
cell
emetine resistance gene: analysis of cDNA and genomic sequences encoding ribosomal protein S14. J. Biol. Chem. 258 (1983) 13236-13242. Nieuwint, R.T.M.,
Molenaar,
C.M.T.,
Van
Bommel,
J.H.,
Van
Raamsdonk-Duin, M.M.C., Mager, W.H. and Planta, R.J.: The gene for yeast ribosomal protein S31 contains an intron in the leader sequence.
Curr. Genet.
10 (1985) 1-5.
Nomura,
M., Gourse,
R. and Baughman,
of ribosomes and ribosomal (1984) 75-l 17. Rhoads,
G.: Regulation
components.
D.D., Dixit, A. and Roufa,
D.J.: Primary
structure
ribosomal protein S14 and the gene that encodes 6 (1986) 2774-2783. Sanger,
F., Nicklen,
terminating
S. and Coulson,
inhibitors.
5463-5461. Suzuki, K., Olvera,
Proc.
J. and Wool,
of the synthesis
Annu. Rev. Biochem.
Acad.
Sci.
LG.: The primary
of human
it. Mol. Cell. Biol.
A.R.: DNA sequencing Natl.
USA
with chain74 (1977)
structure
of rat
ribosomal protein L9. Gene 93 (1990) 297-300. Tedder, T.F., Strueli, M., Schlossman, S.F. and Saito, H.: Isolation structure of a cDNA encoding the B 1 (CD20) cell-surface human B lymphocytes. Proc. Natl. Acad. Sci. USA 208-211. Towbin, H., Staehelin,
T. and Gordon,
53
J.: Electrophoretic
and
antigen of 85 (1988) transfer
of
proteins from polyacrylamide gels to nitrocellulose sheet: procedure and some applications. Proc. Natl. Acad. Sci. USA 76 (1979) 4350-4354.
