Characterization of cDNA clones encoding the human homologue of Saccharomyces cerevisiae ribosomal protein L30

Gene, 123 (1993) 283-285 0 1993 Elsevier Science Publishers

B.V. All rights reserved.

283

0378-l 119/93/$06.00

GENE 06849

Characterization

of cDNA clones encoding the human homologue of Saccharomyces cerevisiae ribosomal protein L30

(Yeast; Xenopus laevis; ribosomes; cloning)

Keith R. Johnson Department of Biology, University of Toledo, Toledo, OH 43606, USA Received by W.M. Holmes:

16 May 1992; Accepted:

15 June 1992; Received at publishers:

15 September

1992

SUMMARY

We have isolated cDNA clones encoding the human homologue (hL30) of yeast ribosomal protein (r-protein) L30. The hL30 nucleotide (nt) sequence shows high homology to the yeast sequences and also to a partial Xenopus laevis sequence previously identified as an immunoglobulin heavy chain. The 5’ end of hL30 is pyrimidine-rich, as is the case for most other mammalian r-protein mRNAs. The open reading frame consists of 157 codons with a C-terminal region that is different from corresponding regions of the yeast proteins. In several human tissue culture cells, the mRNA encoding hL30 is approx. 700 nt in length.

INTRODUCTION

Eukaryotic cytoplasmic ribosomes are composed of RNA and protein components. The RNAs are derived from the transcriptional products of both RNA polymerases I and III. The proteins are translated from some 80 different messenger RNAs (Wool et al., 1990). The aa sequences of a number of the r-proteins are known, either from direct determination or deduced from corresponding cDNAs. It is thought that each r-protein is synthesized in the cytoplasm, translocated to the nucleolus (Meier and Blobel, 1990; Roberts, 1989) and, in a directed fashion, becomes part of the growing ribosome (Tollervey and Hurt, 1990). Nuclear ribosomes are apparently not

Correspondence to: Dr. K.R. Johnson, Department of Biology, University of Toledo, Toledo, OH 43606, USA. Tel. (419)537-4919; Fax (419)537-7737. Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; hL30, human homologue of yL30; hL30, gene encoding hL30; kb, kilobase or 1000 bp; nt, nucleotide(s); ORF, open reading frame; r, ribosomal; UTR, untranslated region; X., Xenopus; yL30, yeast L30 r-protein; yL30, gene encoding yL30.

capable of protein synthesis but must mature in the cytoplasm following exit from the nucleus. In this report, we describe the characterization of cDNA clones encoding hL30, the human homologue of Saccharomyces cerevisiae r-protein L30 (yL30 was previously called RP29; Mitra and Warner, 1984). While conducting homology searches, we identified a partial clone previously thought to encode an X. laevis immunoglobulin heavy chain (Brown et al., 1981) that is probably the yL30 homologue in this species. These data add to the available sequences of mammalian r-proteins.

EXPERIMENTAL

AND DISCUSSION

(a) Sequence of hL30 The original cDNA clone was identified by chance during hybridization screening of a JAR human choriocarcinoma (Patti110 et al., 1971) cDNA library in pUC19 (Yanisch-Perron et al., 1985). Additional clones were isolated by hybridization (Sambrook et al., 1989) from a WI38 cell (normal human diploid fibroblast; Hayflick and Moorhead, 1961) cDNA library in Agtl 1 (Clontech, Palo

284

Alto, CA). Selected restriction fragments were subcloned into pUC vectors and sequenced (Johnson, 1990). The sequence presented in Fig. 1 was determined from the longest clone isolated from the WI-38 cell Agtl 1 library and was completely determined on both strands. The sequence contains 39 nt predicted to be 5’ UTR, a 471-nt ORF and a 46-nt 3’ UTR. The extreme 5’ end contains a pyrimidine-rich sequence; 23 of the first 25 nt are C or T. This feature is typical of mRNAs encoding mammalian r-proteins (Hariharan and Perry, 1990; Zahradka et al., 1990). The predicted start codon is the first ATG in the sequence and it inaugurates a 157-codon ORF. This ATG lies in a sequence favorable for translation initiation (reviewed in Kaufman, 1990). The ORF encodes a protein of 17 758 Da and p1 of 9.6. (b) Comparison

of deduced aa sequences

There are two yL30 genes (Baronas-Lowell and Warner, 1990; Mitra and Warner, 1984) that are predicted to encode almost identical proteins (Fig. 2). The deduced aa sequence of hL30 shows regions of high homology to the yL30 sequences (Fig. 2). Between yL30A and hL30,71 of the first 124 aa are identical. A number of the substitutions are conservative; for example, there are seven instances where Arg and Lys are interchanged. The two sequences agree especially well in the N-terminal portion

TTTTTTCGC

CAC H

GGG AGG G R

CGC TAC GCC AGG R Y A R

ACC GAC GGG AX T D G K"

AAT N

GCG AAA A K

TGC

TX F

GAA E

GAG E

c

TGG ACT w TV

GA?+ ATT E I

CM Q

CTG L

GAG E

TGC

RGT

c

s

TCG

s

GCT A

TTT F

AGC

CTTTCCGTGG

AAG K

ATA AAC I N

GTC ”

CATCTTTTGT

ATG M

s

GGG TAC G Y

cm

TCC

L

s

AN.? K

AAG K

ATC I

AGCTGTCGCC

TAC Y

CCC P

GGA G

39

87 16

GTT TTC CAG TTT CTT F Q F L

135 32

AGG R

AAT N

CCT P

CGG CAG R Q

183

231 64

48

GTC CTC TX L Y

AGA AGG AAG R R K

CAC AAA H K

AAG K

GGA CAG TCG G Q S

RAG K

ACC T

GCA A

AAA K

TTC F

CAG Q

?.GG 279 R 80

AAA K

AGA R

CGC R

CGA R

GTC ”

GCC ATT ACT A I T

GGT GCA XT G A S

CTT GCT GAT ATA ATG GCC ?.AG AGG I. A D I M A K R

AAT N

CAG Q

327 96

Au. K

GCT A

GCT A

375 112

GCA ATG

CCT P

GAA E

GTT ”

AGA R

K

AAG

GCT A

CAR Q

CGA R

GA.& CAA E 0

GCT A

AK I

AGG R

A.&G GAA

RAG K

CM, 0

GCA A

TCT

AAA K

AAG K

ACT T

A

M

GCT A

423 128

GCR A

GCA R

cm

AAG K

CAA 0

AAG K

ATT I

GTG ”

RAG K

471 144

GTT

K

E

GCA A

AAA K

AAG K

GCT A

GCT A.

GCT A

AAG K

GCR A

cm

AC?+ RAG T K

CCT

GTG

AU,

GTT

TCA

GCT

CCC

CGA

S

A

P

R"

P"

K"

ACTGGCAGAT

P

TAGATTTTTA

-GATT

s P GGT

G

GGA AAA G K

GGATTATAAC

CGC TAA R l **

TCTLA,lr

513 157 556

Fig. 1. The nt sequence of hL.30 and deduced aa sequence. Restriction fragments were subcloned into pUC plasmids and sequenced as described (Johnson, 1990). The nt sequence of the longest clone found in the WI-38 cell library is presented. The sequence of the EcoRI linker at the 5’ end is not included. There was no linker at the 3’ end and the sequence is terminated after the poly(dA) tail. The sequence is broken into codons in the predicted ORF with the aa given in single-letter code beneath the codons. The polyadenylation signal is underlined in the 3’ UTR region. Asterisks indicate the stop codon. The nt sequence has been assigned the accession No. M94314 in the GenBank/EMBL/ DDJB databases.

YKIYPGHGRR A....*R*TL A""'R'TL

YARTDGKVTQ F"'G'S.1.R FV'G'S'I'R

FLNAKCESAF 'Q'S.SA'L' 'Q'S'SA'L'

LSKRNPRQIN KQRK"'R'A KQRK.**R*A

50 50 50

KGQSEEIQKK

RTRRAVKFQR

AITGASLADI

.S.KT..A.. .S.KT..A"

P......DL. P......DL.

MAKRNQKPEV . . . ..S" KER'SL.... KER.SL....

100 S*

..IT..VA.. ..IT.."A.. AAKEAKKAKQ . . ..V....K

ASKKTAMA-A

AKAPTKAAPK

QKIVKP--VK

. ..A..__..

ss*

'N"K*"EK .N.*K'R*EK

'AR'AEK'KS 'AR*AEK'KS

*GTQSSKFS. 'GVQGSKVS'

*Q-A.GAFQ. .Q-A.GAFQ.

149 149

. . ..__~_p

v.v.s..rv..

100 100 141

157 63* 155 155

Fig. 2. Comparison of four deduced aa sequences. The deduced aa sequences for r-proteins L30 are shown, In each case, the entire aa sequence deduced from the ORF is included. The numbers at the right of each sequence correspond to the cumulative number of aa. The reference sequence is that of hL30 (H) and is numbered as in Fig. I. The X. laeuis sequence (Xe) is deduced from the nt sequence of Brown et al. (1981). The asterisk adjacent to the aa number of the X. laeois sequence indicates that it is a partial sequence. The yeast aa sequences (Y30A and Y30B) are taken from the gene sequences of Mitra and Warner (1984) and Baronas-Lowell and Warner (l990), respectively. Identities with the reference

sequence

are indicated

with a heavy dot. Gaps have

been inserted in the C-terminal region to increase the alignment of similar residues. Homology searches used the algorithms of Pearson and Lipman (1988) and were conducted using the electronic mail services of the Intelligenetics computer facilities. Further C-terminal ends was done manually.

alignment

at the

while there is less agreement between the C-terminal sequences. The functional consequences of these differences are unknown. In addition to the yeast sequences, the homology search against the nt data bases revealed a significant score against a sequence described as a partial clone for X. laeuis immunoglobulin heavy chain (Brown et al., 1981). If this nt sequence is translated in a reading frame different from that chosen by the authors, the aa sequence labeled Xe in Fig. 2 is obtained. This ORF shows significant homology to the predicted human and yeast sequences. The agreement between the predicted X. laevis, yeast and human proteins suggests that the X. laevis sequence is that of the corresponding r-protein. In contrast to yL30A and B, the similarity between the predicted X. laevis and human sequences extends to the stop codon in hL30 (Fig. 2). It is apparent from these data that the C-terminal ends of the yeast and vertebrate homologues are quite different from one another. Approximately 5 pg of total RNA from JAR cells, HL60 cells (Gallagher et al., 1979) and RPM18402 cells (Huang et al., 1974) were run on denaturating gels (Fourney et al., 1988) and probed with the full-length cDNA encoding hL30. All three cell lines gave identical signals; each sample showed one hybridizing band of about 700 nt (not shown).

285 (c) Conclusions In conclusion, we have characterized cDNA clones that represent the human homologue of yeast r-protein L30. We found evidence for only one type of transcript encoding this protein in human tissue-culture cell lines. We have tentatively identified a previously published sequence (Brown et al., 1981) as the X. laeuis homologue of L30. This sequence adds to the growing data base of r-protein sequences.

somal protein

promoter:

I wish to acknowledge the technical assistance of Ms. Rebecca Castle and helpful discussions with Dr. Margaret Wheelock. This work was supported by grant NIH GM41 116 and the Ohio Board of Regents.

of the polypyrimidine

initiator

87 (1990) 1526-1530. Hayflick, L. and Moorhead, P.S.: The serial cultivation of human diploid cell strains. Exp. Cell Res. 25 (1961) 585-621. Huang, CC., Hou, Y. Woods, L.K., Moore, GE. and Minowada, J.: Cytogenetic study of human lymphoid T-cell lines derived from lymphocytic leukemia. J. Natl. Cancer Inst. 53 (1974) 655-660. Johnson, K.R.: A small-scale plasmid preparation yielding DNA suitable for double-stranded sequencing and in vitro transcription. Anal. Biochem. 190 (I 990) 170- 174. Kaufman, R.J.: Control of translation Setlow, J.K. (Ed.), Genetic 1990, pp. 243-273.

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