Evolutionary conservation of ribosomal protein mRNA sequences: application for expansion of corresponding cDNA and gene libraries

Bioehimiva et Biophvsica Acta 825 (1985) 393-397 Elsevier

393

BBA 91497

Evolutionary conservation of ribosomal protein m R N A sequences: application for e x p a n s i o n of c o r r e s p o n d i n g c D N A and g e n e libraries Oded Meyuhas Developmental Biochemistry Research Unit, Institute of Biochemisto', The Hebrew University and Hadassah Medical School P.O. Box 1172, Jerusalem 91010 (Israel) (Received February 18th, 1985) (Revised manuscript received June l I th, 1985)

Key words: Ribosomal protein; Evolutionary conservation; mRNA sequence; Nucleic acid hybridization: Gene library

Cloned cDNAs, containing ribosomal protein sequences from mouse (five cDNAs) or Xenopus laevis (six cDNAs), were used to estimate the evolutionary conservation, from insects to mammals, of the corresponding mRNA sequences. Northern blot analysis reveals a variable degree of homology between these sequences in different eukaryotes. Thus, among the ribosomal protein cDNA clones utilized, some exhibit complete, others partial, and a few no interphyla cross-hybridization. Melting profile analysis was employed to quantitate this homology. It is proposed that for expansion of eukaryotic ribosomal cDNA and gene libraries, one can exploit the interspecies homology of the corresponding sequences. However, the diverse evolutionary conservation of individual ribosomal protein gene sequences should be taken into account.

Introduction The ribosomal apparatus is generally regarded as highly resistant to evolutionary alterations. Thus, the eukaryotic ribosomal RNA sequences tend to be highly conserved over a wide range of species [1-4]. A similarity in the electrophoretic properties of ribosomal proteins among different eukaryotes [5-7] suggests that there is some evolutionary conservation of these proteins. Interspecies homology, at the nucleic acid level, has only been reported for mammalian rp- (ribosomal protein) sequences [8-11]. During the recent years rpcDNA libraries of various higher eukaryotes have been constructed and subsequently the corresponding genomic clones were isolated. These were derived from Drosophila [12,13], Xenopus [14], mouse [8] and Chinese hamster [15]. For all these species the repertoire of cloned rp- sequences is limited and far from complete. Study of the regulatory mechanisms ensuring coordinate synthesis

of all ribosomal proteins and structural analysis of these proteins through the corresponding DNA sequences require cDNA and genes of additional ribosomal proteins. Thus, a demonstration of the conservation of the sequence of mRNAs and, hence, of the genes encoding for ribosomal proteins, between evolutionary remote species, is of a practical importance. However, as reported herein, interphyla homology ranging from insects to mammals is maintained by only some rp-mRNAs. Others exhibit detectable sequence conservation of more limited character, such as from insects to amphibia or from amphibia to mammals, while several exhibit homology only within the same phylum.

Materials and Methods Total RNA from Drosophila melanogaster was extracted according to Chirgwin et al. [16] and from human epidermoid carcinoma according to

0167-4781/85/$03.30 .~ 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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Kirby [17]. Cytoplasmic RNA from livers of mouse, rat or Xenopus was extracted as described by Schibler et al. [18]. Poly(A) + R N A (messenger RNA containing poly(A) at its 3' end) was isolated according to Schibler et al. [19]. Methods for gel electrophoresis of RNA, direct immobilization of R N A on nitrocellulose filter and blot hybridization have been described in a previous publication from this laboratory [11]. RNA samples were sizefractionated by electrophoresis on 1.2% agarose gels containing formaldehyde, blotted onto nitrocellulose filters and hybridized for 42 h with )~Pnick-translated probes at 42°C in a solution containing 50% formamide and 5 × SSC (1 × SSC = 150 mM NaC1, 15 mM sodium citrate). Blots were washed for 60 min in 0.5 1 × SSC, 0.1% SDS (sodium dodecyl sulfate) at 42°C and then autoradiographed. For measurement of the thermal stability of hybrids poly(A) ) RNA was dot-blotted on nitrocellulose squares (1 cm 2) and hybridized with 32P-labelled rp-cDNA clone. After washing at 35°C for 30 min in 1 × SSC, filters were incubated

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for 30 min in 0.1 × SSC, 0.1% SDS at various temperatures ranging from 35°C to 75°C. The squares were then washed in 0.1 × SSC at room temperature and the radioactivity retaining on the filters was measured. Results and Discussion

In previous papers we described the cloning of several mouse rp-cDNAs [8] and the use of these recombinant DNAs in demonstrating the homology between rp- sequences in different mammals [9 11]. In order to examine the range of rp- sequence conservation throughout the evolution of eukaryotes, mRNAs from various species were size-fractionated, transferred to nitrocellulose filter and hybridized with mouse rp-cDNA clones (Fig. 1). When ribosomal protein L18 eDNA was used as a probe, a major hybridization band was detected in poly(A) + total R N A (total cellular RNA containing poly(A) at its 3' end) isolated from Drosophila melanogaster, and human epider-

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Fig. 1. E l e c t r o p h o r e t i c a n a l y s i s of r p - m R N A s f r o m v a r i o u s species. R N A s a m p l e s were s i z e - f r a c t i o n a t e d on 1.2% a g a r o s e gel c o n t a i n i n g 2.2 M f o r m a l d e h y d e , t r a n s f e r r e d to n i t r o c e l l u l o s e filter a n d h i b r i d i z e d with ~2 P - n i c k - t r a n s l a t e d . m o u s e r p - c D N A clones. LIB lanes: 5 ,ag of p o l y ( A ) ~ total R N A f r o m D. mehmoga~'ter (D) a n d f r o m h u m a n e p i d e r m o i d c a r c i n o m a ( H ) (nitrocellulose filter c a r r y i n g these t w o R N A s was k i n d l y p r o v i d e d b y H. S o r e q f r o m the W e i z m a n n Institute of Science) a n d 5 ~tg of liver p o l y ( A ) ' m R N A f r o m X. lae¢,is (X). rat (R) a n d m o u s e (M) were h y b r i d i z e d with m o u s e E l 8 e D N A clone: L19 lanes: 5 p.g of Drosophila p o l y ( A ) + total R N A ( D ) a n d 5 /xg of liver p o l y ( A ) ' m R N A f r o m X. laet,i.~ (X), rat (R) a n d m o u s e (M) were h y b r i d i z e d with m o u s e k 1 9 e D N A clone; L7 lanes: 6 /xg of Drosophila p o l y ( A ) ~ total R N A (D) a n d 6 p.g of liver p o l y ( A ) ) m R N A f r o m Xenopus (X) a n d m o u s e (M) were h y b r i d i z e d with m o u s e L7 e D N A clone: S16 lanes: as for L7 lanes but R N A s w e r e h y b r i d i z e d with S16 e D N A clone. T h e p o s i t i o n s o f m o u s e 28 S a n d 18 S a n d of E.coh 23 S a n d 16 S r i b o s o m a l R N A s were d e t e r m i n e d in parallel t r a c k s a f t e r s t a i n i n g with 2 # g / m l e t h i d i u m b r o m i d e .

395

moid carcinoma and in p o l y ( A ) + m R N A from livers of Xenopus laevis, mouse and rat (Fig. 1, L18). Likewise, mouse L19 c D N A also exhibits sequence conservation from Drosophila to mammals of the corresponding m R N A (Fig. 1, L19). The sizes of L18 and L19 m R N A s in all eukaryotes examined are similar (740 and 910 nucleotides, respectively). However, despite this wide range of interphyla homology, no cross-hybridization with yeast poly(A) R N A has been observed (data not shown). Thermal-stability analysis was employed to quantitate the sequence homology between mouse L18 m R N A and that of Xenopus and Drosophiht. Poly(A) + m R N A from livers of mouse or Xenopus and poly(A) + total RNA from Drosophila were immobilized on nitrocellulose filter and hybridized to 32p-nick-translated mouse L18 cDNA. Filters were washed at increasing temperatures as described in Materials and Methods (Fig. 2). The resulting melting profiles reveal l l ° C and 23°C differences between t m (melting temperature) of

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Fig. 2. T h e r m a l stability of hybrids of m o u s e r p - c D N A with r p - m R N A s from various species. 2 /*g of mouse liver poly(A) + m R N A (e), 6 ,ug of poly(A) + m R N A from X e n o p u s liver ( I ) a n d 6 /*g poly(A) + total R N A from Drosophila (*) were i m m o b i l i z e d on nitrocellulose squares a n d hybridized with the i n d i c a t e d 32p-nick t r a n s l a t e d r p - c D N A clones. The filters were w a s h e d as described in Materials and Methods. The radioactivity retained on the filter was m e a s u r e d and from that value the released radioactivity was calculated and plotted. 6 ktg of E.coli t R N A i m m o b i l i z e d on nitrocellulose squares served as a control for nonspecific b i n d i n g and retention of radioactivity t h r o u g h o u t the experiment.

hybrids involving mouse poly(A) + m R N A (tm = 67.5°C) and those involving Xenopus poly(A) + m R N A ( t m = 5 6 . 5 ° C ) , or Drosophila poly(A) + total R N A (tm = 44.5°C), respectively. Assuming that a A t m of 1°C results from about 1.5% mismatched basepairs [20], the decreased t m values of hybrids involving Xenopus or Drosophila L18 m R N A reflect about 16.5% and 34.5%, respectively, of sequence diversity, when compared to the corresponding mouse sequence. Similar melting curves have been observed with mouse L32 c D N A hybridized with mouse poly(A) ~ m R N A (tin= 68.5°C), Xenopus poly(A) + m R N A (tin= 54°C) and Drosophila poly(A) + total RNA ( T m = 46.5°C) (Fig. 2), indicating a comparable evolutionary drift of L32 and L18 sequences from insects to mammals. However, this evolutionary conservation is not universal for all r-protein sequences. Mouse L7 c D N A exhibits homology with the corresponding amphibian sequence but not with that of Drosophila (Fig. 1, L7). The mouse $16 c D N A shares no homologous sequences even with the amphibian m R N A under the relatively low stringent conditions used for washing the blot (1 × SSC, 42°C) (Fig. 1, S16). The lack of detectable hybridization with the Drosophila or Xenopus m R N A s cannot be attributed to the quality of these RNA preparations, since the LI 8 m R N A bands of these species were apparent when this blot was hybridized with L18 probe (data not shown). Similar variability of sequence conservation has been observed when Xenopus rp-cDNAs [14] were hybridized with mouse poly(A) + m R N A or Drosophila poly(A) + total RNA (Table I). When Xenopus L1 c D N A clone (the nomenclature of Xenopus ribosomal protein is not related to that of mammals) was used as a probe, we could not detect any hybridizable sequence in either the fly or the murine m R N A , even under the relatively low stringent conditions used in this experiment. However, under these conditions, two Xenopus rp-cDNA clones (L14 and S1) hybridized to the Drosophila m R N A and an additional two (L32 and $8) to mouse m R N A . Of the amphibian rpc D N A clones examined, only one (S19) hybridized to the corresponding m R N A of both the insect and the mammal. It is worth noting that, based on hybridization analysis (data not shown), none of these amphibian rp-cDNAs corresponds to any of

396 "FABLE I SIZE OF MOUSE A N D DROSOPHILA mRNA SEQUENCES W H I C H WERE DETECTED BY XENOPUS RIBOSOMAL PROTEIN cDNA CLONES 5 p,g of poly(A) + mRNA from mouse fetuses or poly(A) + total RNA from D. rnelanogaster were size-fractionated, transferred to nitrocellulose filters and hybridized as described in Materials and Methods. Filters were washed in l ×SSC, 0.1% SDS at 42°C. (Nitrocellulose filter carrying the Drosophila RNA used in this experiment was kindly provided by Z. Lev from Technion-lsrael Institute of Technology.) n.d., not detected.

Xenopus cDNA clone "

Xenopus ribosomal protein sequence

Size of hybridizable mRNA (bases)

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1500 750 550 1000 850 650

n.d. n.d. 520 n.d. 740 640

n.d. 730 n.d. 950 n.d. 6l0

102 92 78 91 62 69

" The X. laevis rp-cDNA clones were obtained from F. Amaldi [14]. b Data for the size of X. laevis rp-mRNAs from Pierandrei-Amaldi et al. [21].

the mouse rp- probes used in this study. The reason for the variable degree of conservation of r p - m R N A sequence is not known. However, the observed divergence is not necessarily indicative of variation in the primary structure of the protein. Both the degeneracy of the genetic code and the presence of 3' and 5' untranslated regions in the eukaryotic m R N A s permit more than 30% variation in the m R N A base composition without altering the primary structure of the protein encoded by this mRNA. It is noteworthy that in all cases of detectable interphyla homology, with the exception of Xenopus $8 sequence, there is a considerable conservation of the r p - m R N A size (Fig. 1 and Table I). Obviously, comparative sequence analysis of the m R N A from these species should enable us to determine whether the observed different melting profiles also represent variation in the amino acid sequence, and to what extent the untranslated, regions are similarly conserved. In an attempt to expand the present collections of rp-cDNAs and the corresponding genomic clones, one can take advantage of the interspecies homology of r p - m R N A sequences and utilize available rp- probes, even from evolutionary distant species, to isolate additional rp- clones. However, two facts should be taken into account while using heterologous rp- probes: (a) the interspecies

cross-hybridization of rp- sequences is not universal; (b) the stability of rp- hybrids is variable and depends on the evolutionary divergence of the participating sequences. The proposed approach should save, at least for several ribosomal proteins, the tedious work otherwise required for identification of rp- sequences by hybrid selection of the corresponding m R N A s [8].

Acknowledgement The author wishes to thank Dr. F. Amaldi from the University of Rome for his generosity in providing the Xenopus laevis ribosomal protein c D N A clones. This research was supported by a grant from the Israel National Academy of Sciences and Humanities--Basic Research Foundation. O.M. is a ' Bat-Sheva Fellow'.

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