Short
Communication
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Acta Biochim Biophys
Sin 2005,37:573-579
doi:10.1111/j.1745-7270.2005.00072.x
Ribosomal Protein Genes S23 and L35 from Amphioxus Branchiostoma
belcheri tsingtauense: Identification and Copy Number
Xian LI, Shi-Cui ZHANG*,
Zhen-Hui LIU, and Hong-Yan LI
Department of
Marine Biology, Ocean University of China, Qingdao 266003, China
Received: January
28, 2005
Accepted: May 18,
2005
This work was
supported by a grant from the National Natural Science Foundation of China
(No. 30470203)
*Corresponding
author: Tel, 86-532-2032787; Fax, 86-532-2032787; E-mail, [email protected]
Abstract The complete cDNA and
deduced amino acid sequences of the ribosomal proteins S23 (AmphiS23) and L35
(AmphiL35) from amphioxus Branchiostoma belcheri tsingtauense were
identified in this study. AmphiS23 cDNA is 546 bp long and encodes a protein of
143 amino acids. It has a predicted molecular mass of 15,851 Da and a pI of
10.7. AmphiL35 cDNA comprises 473 bp, and codes for a protein of 123 amino
acids with a predicted molecular mass of 14,543 Da and a pI of 10.8. AmphiS23
shares more than 83% identity with its homologues in the vertebrates and more
than 84% identity with those in the invertebrates. AmphiL35 is more than 63%
identical to its counterparts in the vertebrates and more than 52% identical to
those in the invertebrates. Southern blot analysis demonstrated the existence
of 1–2 copies of the S23 gene and 2–3 copies of the L35 gene in the genome of amphioxus B. belcheri
tsingtauense. This is in sharp contrast to the presence of 6–13 copies of the S23 gene and 15–17 copies of the L35 gene in the rat genome. It is clear that the
housekeeping genes like S23 and L35 underwent a large-scale duplication in the
vertebrate lineage, reinforcing the gene/genome duplication hypothesis.
Key words amphioxus; ribosomal
protein; S23; L35; copy number
Ribosomes are the RNA-protein organelles
that catalyze the sequential addition of amino acids to the carboxyl end of
the growing polypeptide chain, according to the blueprints encoded by mRNA [1].
Each ribosome comprises two subunits: a large (L) and a small (S) subunit. In
eukaryotes, the large 60S subunit is composed of three ribosomal RNAs (rRNAs)
and nearly 50 ribosomal proteins, whereas the small 40S subunit consists of one
rRNA and approximately 30 proteins [2]. Ribosomal proteins are highly
conserved proteins encoded by the housekeeping genes, as their activity is
required for the growth and maintenance of all cell types [3]. Information
contained in the sequences of ribosomal proteins can contribute to unraveling
their evolution and function.The eukaryotic ribosomal protein S23, known as S12 in bacteria and
as either S12 or S23 in Archaea [4], appears to be involved in the
translation initiation step of protein synthesis [5]. The ribosomal protein L35
is found to bind to both initiator and elongator tRNAs [6,7]. The gene encoding
S23 has been identified in several organisms such as mammals (GenBank accession
No. AAS55902 for Chinchilla lanigera; AAH70221 for Homo sapiens;
CAA54584 for Rattus norvegicus; AAS59430 for Sus scrofa),
amphibians [8], teleosts [9], insects (GenBank accession No. AAV34880 for Bombyx
mori; BAD26702 for Plutella xylostella), nematodes [10,11], and
annelids (GenBank accession No. CAC14789 for Lumbricus rubellus). The
gene encoding L35 has been isolated from organisms including mammals [12],
birds [13], reptiles (GenBank accession No. AAR10441 for Ophiophagus hannah),
amphibians [12], teleosts [14,15], insects (GenBank accession No. AAV34846 for Bombyx
mori), and nematodes (GenBank accession No. AAA28216 for Caenorhabditis
elegans). Amphioxus or lancelet, a basal chordate, has been widely known as
the living fossil most closely related to the proximate ancestor of
vertebrates in phylogeny [16,17]. Liu et al. [18,19] recently reported
the cloning of ribosomal proteins S15a, L19, S20 and L10 cDNAs from amphioxus Branchiostoma
belcheri tsingtauense. However, no information has been available so far
for S23 and L35 in this evolutionarily important organism. Gene/genome duplication has been an interesting topic for biologists
for decades [20–22]. It is proposed that
two rounds of large-scale gene duplication took place during early chordate
evolution: one occurred close to the origin of vertebrates, the other close to
the origin of jawed vertebrates [21,23–25].
Comparison of the numbers of luxury protein genes such as Hox [26], Otx
[27], Msx [28] and hedgehog [29] provides substantial evidence
for this hypothesis. Evolutionarily, it remains uncertain whether the
housekeeping genes like S23 and L35 also follow the two-round
duplication rule, and data comparing housekeeping gene copy numbers in
different species are still lacking. The aims of the present study were to characterize S23 and L35 cDNAs
from amphioxus B. belcheri tsingtauense and to determine these gene copy
numbers in its genome.
Experimental Procedures
The cDNA library was constructed using the SMART cDNA library
construction kit (Clontech, Palo Alto, USA) according to the method described
previously [30]. cDNA clones were randomly selected for sequencing. Both strands
of all selected clones were sequenced with the ABI PRISM 377XL DNA sequencer
(PE company, Foster City, California, USA ) and all sequences were then
analyzed for coding probability with the DNATools program developed by Rehm
[31].Initial comparison against the GenBank protein database was
performed using the BLAST network server at the National Center for
Biotechnology Information [32]. Multiple protein sequences were aligned by the
Clustal method, using the MegAlign program in the DNAStar software package
developed by Burland [33]. Accession numbers of the ribosomal protein sequences
in the GenBank database used for comparison are listed in Table 1 and Table
2.Genomic DNAs for Southern blotting analysis were isolated from adult
amphioxus. A total of 30 amphioxus were ground in liquid nitrogen, and the
powder was suspended in 15 ml of lysis buffer (pH 8.0) containing 10 mM
Tris-HCl, 100 mM EDTA and 0.5% SDS. After treatment with proteinase K (100
mg/ml, final concentration) at 55 ?C for 3 h, it was cooled to room temperature
and mixed with an equal volume of saturated phenol (pH 8.0). The mixture was
centrifuged at 5000 g at 4 ?C for 20 min, and the supernatant was pooled
and mixed with an equal volume of phenol:chloroform (1:1, V/V).
The mixture was centrifuged as above and the supernatant was collected. DNA
was precipitated by ethanol and digested with various restriction enzymes at 37
?C for 20 h: EcoRV, PstI, HindIII, BstXI and BglII
(one unit per microgram DNA) for genomic DNA to be hybridized with digoxigenin
(DIG)-labeled cDNA probes of AmphiS23; and EcoRI, PstI, HindIII,
EcoRV and BstXI (one unit per microgram DNA) for genomic
DNA to be hybridized with DIG-labeled cDNA probes of AmphiL35. The
digested DNAs were separated on a 1% agarose gel using 1?TBE (89 mM Tris-borate and 2 mM EDTA) and transferred onto nylon
membranes (Osmonics Inc., Minnesota, USA). The membranes were hybridized with
the DIG-labeled DNA probes produced with a DIG DNA labeling kit (Roche, Basel,
Switzerland). Hybridized bands were visualized according to the instructions
of the detection kit.
Results and Discussion
The first cDNA encoding amphioxus ribosomal protein S23, AmphiS23,
was identified from the gut cDNA library as revealed by BLAST search. Fig.
1 shows the nucleotide and deduced amino acid sequences of AmphiS23 cDNA
(GenBank accession No. AY168453). It was 546 bp long and consisted of a 26 bp 5‘
untranslated region (UTR), an open reading frame (ORF) of 432 bp and an 88 bp 3‘
UTR. The ORF encoded a 143 amino acid protein with a calculated molecular mass
of 15,851 Da and a pI of 10.706. The 5‘ UTR had an in-frame stop codon
TGA upstream of the first start codon ATG and a polypyrimidine sequence, CTTTC,
which has been found at the 5‘ end of many eukaryotic ribosomal protein
mRNAs [34]. The 3‘ UTR had a polyadenylation signal AATAA 18 bases
upstream of the poly(A) site which is required for post-translational
cleavage-polyadenylation of the 3‘ end of the pre-mRNA [35]. The deduced protein sequence of AmphiS23 was compared with those of
the other known S23 proteins from various organisms in the GenBank database (Table
1). AmphiS23 shares more than 83% identity with its homologues in the
vertebrates such as humans, rats, pigs, frogs and teleosts, and more than 84%
identity with those in the invertebrates like insects, annelids and nematodes (Fig.
2).AmphiS23 is a rather hydrophobic protein with 50 hydrophobic amino
acids out of 143 residues. It has a high percentage of basic amino acids (20
lysines and 13 arginines) mostly located in the N-terminal half of the deduced
amino acid sequence, and a low percentage of acidic amino acids (5 aspartic
acids and 7 glutamic acids) mostly situated in the C-terminal half. The strong
basic character of S23 including AmphiS23 may be instrumental for its binding
to rRNA in the 40S subunit of eukaryotic ribosomes [36–38]. The second identified cDNA clone encoded amphioxus ribosomal protein
L35, AmphiL35. Fig. 3 shows the nucleotide and deduced amino acid
sequences of AmphiL35 cDNA (GenBank accession No. AY168767). The cDNA comprised
473 bp and included a 5‘ UTR of 27 bp, an ORF of 372 bp and a 3‘
UTR of 74 bp. The ORF encoded a 123 amino acid protein with a calculated
molecular mass of 14,543 Da and a pI of 10.8. The 5‘ UTR of AmphiL35 had
an oligopyrimidine tract CTTTTTCC upstream of the start codon ATG, which
consists of a C residue at the cap site, followed by an uninterrupted sequence
of up to 13 pyrimidines [39–43], and
possibly plays a critical role in the translational control mechanism [44]. The
3‘ UTR of AmphiL35 had a polyadenylation signal ATTAA, which is required
for post-translational cleavage-polyadenylation of the 3‘ end of the
pre-mRNA.The second identified cDNA clone encoded amphioxus ribosomal protein
L35, AmphiL35. Fig. 3 shows the nucleotide and deduced amino acid
sequences of AmphiL35 cDNA (GenBank accession No. AY168767). The cDNA comprised
473 bp and included a 5‘ UTR of 27 bp, an ORF of 372 bp and a 3‘
UTR of 74 bp. The ORF encoded a 123 amino acid protein with a calculated
molecular mass of 14,543 Da and a pI of 10.8. The 5‘ UTR of AmphiL35 had
an oligopyrimidine tract CTTTTTCC upstream of the start codon ATG, which
consists of a C residue at the cap site, followed by an uninterrupted sequence
of up to 13 pyrimidines [39–43], and
possibly plays a critical role in the translational control mechanism [44]. The
3‘ UTR of AmphiL35 had a polyadenylation signal ATTAA, which is required
for post-translational cleavage-polyadenylation of the 3‘ end of the
pre-mRNA.Comparison of the deduced AmphiL35 amino acid sequence with that of
its counterparts in the GenBank database (Table 2) showed that AmphiL35
possesses more than 63% identity with its homologues in the vertebrates
including human beings, pigs, birds, snakes, frogs, catfish, zebrafish and
seahorses, and more than 52% identity to those in the invertebrates like
insects and nematodes. As Fig. 4 shows, like the other known L35
proteins, AmphiL35 also has an excess of basic residues over acidic ones (4:1),
which is the same as the proportion of basic residues versus acidic ones in the
amino acid sequence of L35 from mammals, birds, reptiles, amphibians, teleosts,
insects and nematodes.The homology of AmphiS23 and AmphiL35 to their known counterparts
extends the range of species in which these proteins are highly conserved. This
high conservation of S23 and L35 amino acid sequences in various organisms
including the vertebrates and invertebrates suggests they have been subjected
to strong selective pressure during evolution.To analyze the copy number of AmphiS23 and AmphiL35 genes, the
DIG-labeled cDNA probes of AmphiS23 and AmphiL35 were used to hybridize digests
made from amphioxus genomic DNA with either the restriction enzymes EcoRV,
PstI, HindIII, BstXI and BglII or the enzymes EcoRI,
PstI, HindIII, EcoRV and BstXI. The enzymes used do
not digest AmphiS23 or AmphiL35 cDNA strings. For AmphiS23, there is a single
hybridization band for the genomic DNA digested with each of the enzymes PstI,
HindIII, BstXI and BglII, and two hybridization bands
were observed for the genomic DNA digested with EcoRV [Fig. 5(A)].
For AmphiL35, two hybridization bands for the genomic DNA digested with each of
the enzymes EcoRI, PstI, EcoRV and BstXI, and three
bands for the genomic DNA digested with the enzyme HindIII were
revealed [Fig. 5(B)]. These suggest the presence of 1–2 copies of the S23 gene and 2–3 copies of the L35 gene in the genome of amphioxus B. belcheri
tsingtauense. In contrast, there exist 6–13 copies of the S23 gene and 15–17 copies of the L35 gene in the rat genome [4,45]. From the
comparison of the number of AmphiS23 and AmphiL35 genes with that of rat S23
and L35 genes, it is clear that S23 and L35 genes had undergone extensive
duplication in the vertebrate like rat. It therefore appears that the
divergence of the vertebrate from the common ancestor of
cephalochordate/vertebrate is accompanied by a large-scale duplication of the
housekeeping protein genes such as S23 and L35.
Acknowledgements
The authors thank Dr. Haimanti BHATTACHARYA for her critical reading
of the manuscript.
References
1 Ramakrishnan V, Moore PB. Atomic
structures at last: The ribosome in 2000. Curr Opin Struct Biol 2001, 11: 144–154
2 Kay MA, Jacobs-Lorena M. Developmental
genetics of ribosome synthesis in Drosophila. Trends Genet 1987, 3: 347–351
3 Wool IG. The structure and function
of eukaryotic ribosomes. Annu Rev Biochem 1979, 48: 719–754
4 Kitaoka Y, Olvera J, Wool IG. The
primary structure of rat ribosomal protein S23. Biochem Biophys Res Commun
1994, 202: 314–320
5 McMahon G, Landau JV. Effect of S12
ribosomal mutations on peptide chain elongation in Escherichia coli: A
hydrostatic pressure study. J Bacteriol 1982, 151: 516–520
6 Herzog H, H?fferer L, Schneider R,
Schweiger M. cDNA encoding the human homologue of rat ribosomal protein L35a.
Nucleic Acids Res 1990, 18: 4600
7 Ulbrich N, Wool IG, Ackerman E,
Sigler PB. The identification by affinity chromatography of the rat liver
ribosomal proteins that bind to elongator and initiator transfer ribonucleic
acids. J Biol Chem 1980, 255: 7010–7016
8 Klein SL, Strausberg RL, Wagner L,
Pontius J, Clifton SW, Richardson P. Genetic and genomic tools for Xenopus
research: The NIH Xenopus initiative. Dev Dyn 2002, 225: 384–391
9 Karsi A, Patterson A, Feng J, Liu
Z. Translational machinery of channel catfish. I. A transcriptomic approach to
the analysis of 32 40S ribosomal protein genes and their expression. Gene 2002,
291: 177–186
10 Gregory WF, Blaxter ML, Maizels RM.
Differentially expressed, abundant trans-spliced cDNAs from larval Brugia
malayi. Mol Biochem Parasitol 1997, 87: 85–95
11 Kamath RS, Fraser AG, Dong Y, Poulin G,
Durbin R, Gotta M, Kanapin A et al. Systematic functional analysis of
the Caenorhabditis elegans genome using RNAi. Nature 2003, 421: 231–237
12 Strausberg RL, Feingold EA, Grouse LH,
Derge JG, Klausner RD, Collins FS, Wagner L et al. Generation and
initial analysis of more than 15,000 full-length human and mouse cDNA
sequences. Proc Natl Acad Sci USA 2002, 99: 16899–16903
13 Uechi T, TanakaT, Kenmochi N. A complete
map of the human ribosomal protein genes: Assignment of 80 genes to the
cytogenetic map and implications for human disorders. Genomics 2001, 72: 223–230
14 Golling G, Amsterdam A, Sun Z, Antonelli
M, Maldonado E, Chen W, Burgess S et al. Insertional mutagenesis in
zebrafish rapidly identifies genes essential for early vertebrate development.
Nat Genet 2002, 31: 135–140
15 Patterson A, Karsi A, Feng J, Liu Z.
Translational machinery of channel catfish. II. Complementary DNA and
expression of the complete set of 47 60S ribosomal proteins. Gene 2003, 305:
151–160
16 Stokes MD, Holland ND. The lancelet: also
known as ‘amphioxus’, this curious creature has returned to the limelight as a
player in the phylogenetic history of the vertebrates. Am Sci 1998, 86: 552–560
17 Zhang SC, Yuan JD, Li HY. Amphioxus?model
animal for insights into the origin and evolution of the vertebrates. Chin Bull
Life Sci 2001, 13: 214–218
18 Liu M, Zhang SC, Liu Z, Yuan JD, Xu AL.
Identification of the ribosomal proteins S20 and L10 from the amphioxus Branchiostoma
belcheri tsingtauense. Indian J Marine Sci 2004, 33: 231–237
19 Liu ZH, Zhang SC, Liu M, Xu AL. Identification
of the ribosomal proteins S15a and L19 from the amphioxus Branchiostoma
belcheri tsingtauense. Ophelia 2004, 58: 23–27
20 Ohno S. Evolution by Gene Duplication.
New York: Springer Verlag 1970
21 Meyer A, Schartl M. Gene and genome
duplications in vertebrates: The one-to-four (-to-eight in fish) rule and the
evolution of novel gene functions. Curr Opin Cell Biol 1999, 11: 699–704
22 Sankoff D. Gene and genome duplication.
Curr Opin Genet Dev 2001, 11: 681–684
23 Holland PW, Garcia-Fernandez J, Williams
NA, Sidow A. Gene duplication and the origins of vertebrate development. Dev
Suppl 1994: 125–133
24 Sharman AC, Holland PW. Conservation,
duplication and divergence of developmental genes during chordate evolution.
Netherlands J Zool 1996, 46: 47–67
25 Sidow A. Gen(om)e duplications in the
evolution of early vertebrates. Curr Opin Genet Dev 1996, 6: 715–722
26 Holland PW, Garcia-Fernandez J. Hox
genes, developmental evolution and the origin of vertebrates. Ontogenez 1996,
27: 273–279
27 Williams NA, Holland PW. Molecular
evolution of the brain of chordates. Brain Behav Evol 1998, 52: 177–185
28 Sharman AC, Shimeld SM, Holland PW. An
amphioxus Msx gene expressed predominantly in the dorsal neural tube. Dev
Genes Evol 1999, 209: 260–263
29 Shimeld SM. The evolution of the hedgehog
gene family in chordates: Insights from amphioxus hedgehog. Dev Genes Evol
1999, 209: 40–47
30 Liu ZH, Zhang SC, Yuan JD, Sawant MS, Wei
J, Xu A. Molecular cloning and phylogenetic analysis of AmphiUbf80, a new
member of ubiquitin family from the amphioxus Branchiostoma belcheri
tsingtauense. Curr Sci 2002, 83: 50–53
31 Rehm BH. Bioinformatic tools for
DNA/protein sequence analysis, functional assignment of genes and protein
classification. Appl Microbiol Biotechnol 2001, 57: 579–592
32 Altschul SF, Madden TL, Schaffer AA,
Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: A new
generation of protein database search programs. Nucleic Acids Res 1997, 25:
3389–3402
33 Burland TG. DNASTAR’s Lasergene sequence
analysis software. Methods Mol Biol 2000, 132: 71–91
34 Wool IG, Chan YL, Gl?ck A. Mammalian
ribosomes: The structure and the evolution of the proteins. In: Hershey JWB,
Mathews MB, Sonenberg N eds. Translational Control. New York: Cold Spring
Harbor Laboratory Press 1996, 685–732
35 Proudfoot NJ, Brownlee GG. 3‘
non-coding region sequences in eukaryotic messenger RNA. Nature 1976, 263: 211–214
36 Dudov KP, Perry RP. The gene family
encoding the mouse ribosomal protein L32 contains a uniquely expressed intron-containing
gene and an unmutated processed gene. Cell 1984, 37: 457–468
37 Wiedemann LM, Perry RP. Characterization of the
expressed gene and several processed pseudogenes for the mouse ribosomal
protein L30 gene family. Mol Cell Biol 1984, 4: 2518–2528
38 Ulbrich N, Lin A, Wool IG. Identification
by affinity chromatography of the eukaryotic ribosomal proteins that bind to
5.8S ribosomal ribonucleic acid. J Biol Chem 1979, 254: 8641–8645
39 Amaldi F, Pierandrei-Amaldi P.
Translational regulation of the expression of ribosomal protein genes in Xenopus
laevis. Enzyme 1990, 44: 93–105
40 Colombo P, Fried M. Functional elements
of the ribosomal protein L7a (rpL7a) gene promoter region and their
conservation between mammals and birds. Nucleic Acid Res 1992, 20: 3367–3373
41 Perry RP, Meyuhas O. Translational
control of ribosomal protein production in mammalian cells. Enzyme 1990, 44:
83–92
42 Sugawara A, Shiga K, Takasawa S, Yonekura
H, Yamamoto H, Okamoto H. Sequence of the chicken rig gene encoding ribosomal
protein S15. Gene 1991, 108: 313–314
43 Toku S, Tanaka T. The primary structure
of chicken ribosomal protein L37a. Biochim Biophys Acta 1992, 1132: 88–90
44 Levy S, Avni D, Hariharan N, Perry RP,
Meyuhas O. Oligopyrimidine tract at the 5‘ end of mammalian ribosomal
protein mRNAs is required for their translational control. Proc Natl Acad Sci
USA 1991, 88: 3319–3323
45 Suzuki K, Olvera J, Wool IG. The primary
structure of rat ribosomal protein L35. Biochem Biophys Res Commun 1990, 167:
1377–1382