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1.
U17/snR30 is a ubiquitous snoRNA with two conserved sequence motifs essential for 18S rRNA production
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Saccharomyces cerevisiae snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) required for the processing of 18S rRNA. Here, we show that the previously characterized human, reptilian, amphibian, and fish U17 snoRNAs represent the vertebrate homologues of yeast snR30. We also demonstrate that U17/snR30 is present in the fission yeast Schizosaccharomyces pombe and the unicellular ciliated protozoan Tetrahymena thermophila. Evolutionary comparison revealed that the 3'-terminal hairpins of U17/snR30 snoRNAs contain two highly conserved sequence motifs, the m1 (AUAUUCCUA) and m2 (AAACCAU) elements. Mutation analysis of yeast snR30 demonstrated that the m1 and m2 elements are essential for early cleavages of the 35S pre-rRNA and, consequently, for the production of mature 18S rRNA. The m1 and m2 motifs occupy the opposite strands of an internal loop structure, and they are located invariantly 7 nucleotides upstream from the ACA box of U17/snR30 snoRNAs. U17/snR30 is the first identified box H/ACA snoRNA that possesses an evolutionarily conserved role in the nucleolytic processing of eukaryotic pre-rRNA. 相似文献
2.
18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote‐specific 18S sequences
The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non‐coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of ‘orphan’ H/ACA RNAs participate in pre‐rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 ‘orphan’ H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre‐rRNA processing, two evolutionarily conserved sequence elements in the 3′‐hairpin of snR30 base‐pair with short pre‐rRNA sequences located in the eukaryote‐specific internal region of 18S rRNA. The newly discovered snR30‐18S base‐pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3′‐hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein‐binding site. 相似文献
3.
van Beekvelt CA Kooi EA de Graaff-Vincent M Riet J Venema J Raué HA 《Journal of molecular biology》2000,296(1):7-17
Domain III of Saccharomyces cerevisiae 25 S rRNA contains the recognition site for the primary rRNA-binding ribosomal protein L25, which belongs to the functionally conserved EL23/L25 family of ribosomal proteins. The EL23/L25 binding region is very complex, consisting of several irregular helices held together by long-distance secondary and tertiary interactions. Moreover, it contains the eukaryote-specific V9 (D7a) expansion segment. Functional characterisation of the structural elements of this site by a detailed in vitro and in vivo mutational analysis indicates the presence of two separate regions that are directly involved in L25 binding. In particular, mutation of either of two conserved nucleotides in the loop of helix 49 significantly reduces in vitro L25 binding, thus strongly supporting their role as attachment sites for the r-protein. Two other helices appear to be primarily required for the correct folding of the binding site. Mutations that abolish in vitro binding of L25 block accumulation of 25 S rRNA in vivo because they stall pre-rRNA processing at the level of its immediate precursor, the 27 S(B) pre-rRNA. Surprisingly, several mutations that do not significantly affect L25 binding in vitro cause the same lethal defect in 27 S(B) pre-rRNA processing. Deletion of the V9 expansion segment also leads to under-accumulation of mature 25 S rRNA and a twofold reduction in growth rate. We conclude that an intact domain III, including the V9 expansion segment, is essential for normal processing and assembly of 25 S rRNA. 相似文献
4.
G M Veldman J Klootwijk V C de Regt R J Planta C Branlant A Krol J P Ebel 《Nucleic acids research》1981,9(24):6935-6952
We present the sequence of the 26S rRNA of the yeast Saccharomyces carlsbergensis as inferred from the gene sequence. The molecule is 3393 nucleotides long and consists of 48% G+C; 30 of the 43 methyl groups can be located in the sequence. Starting from the recently proposed structure of E. coli 23S rRNA (see ref. 25) we constructed a secondary structure model for yeast 26S rRNA. This structure is composed of 7 domains closed by long-range base pairings as n the bacterial counterpart. Most domains show considerable conservation of the overall structure; unpaired regions show extended sequence homology and the base-paired regions contain many compensating base pair changes. The extra length of the yeast molecule is due to a number of insertions in most of the domains, particularly in domain II. Domain VI, which is extremely conserved, is probably part of the ribosomal A site. alpha-Sarcin, which apparently inhibits the EF-1 dependent binding of aminoacyl-tRNA, causes a cleavage between position 3025 and 3026 in a conserved loop structure, just outside domain VI. Nearly all of the located methyl groups, like in E. coli, are present in domain II, V and VI and clustered to a certain extent mainly in regions with a strongly conserved primary structure. The only three methyl groups of 26S rRNA which are introduced relatively late during the processing are found in single stranded loops in domain VI very close to positions which have been shown in E. coli 23S rRNA to be at the interface of the ribosome. 相似文献
5.
Ethanolamine phosphoglycerol (EPG) is a protein modification attached exclusively to eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is linked to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote Trypanosoma brucei, only domain III is modified by a single EPG. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but nothing is known about the EPG modifying enzyme(s). By expressing human eEF1A in T. brucei, we now show that EPG attachment to eEF1A is evolutionarily conserved between T. brucei and Homo sapiens. In contrast, S. cerevisiae eEF1A, which has been shown to lack EPG is not modified in T. brucei. Furthermore, we show that eEF1A cannot functionally complement across species when using T. brucei and S. cerevisiae as model organisms. However, functional complementation in yeast can be obtained using eEF1A chimera containing domains II or III from other species. In contrast, yeast domain I is strictly required for functional complementation in S. cerevisiae. 相似文献
6.
Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes. 总被引:4,自引:1,他引:3
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The participation of 18S, 5.8S and 28S ribosomal RNA in subunit association was investigated by chemical modification and primer extension. Derived 40S and 60S ribosomal subunits isolated from mouse Ehrlich ascites cells were reassociated into 80S particles. These ribosomes were treated with dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulfonate to allow specific modification of single strand bases in the rRNAs. The modification pattern in the 80S ribosome was compared to that of the derived ribosomal subunits. Formation of complete 80S ribosomes altered the extent of modification of a limited number of bases in the rRNAs. The majority of these nucleotides were located to phylogenetically conserved regions in the rRNA but the reactivity of some bases in eukaryote specific sequences was also changed. The nucleotides affected by subunit association were clustered in the central and 3'-minor domains of 18S rRNA as well as in domains I, II, IV and V of 5.8/28S rRNA. Most of the bases became less accessible to modification in the 80S ribosome, suggesting that these bases were involved in subunit interaction. Three regions of the rRNAs, the central domain of 18S rRNA, 5.8S rRNA and domain V in 28S rRNA, contained bases that showed increased accessibility for modification after subunit association. The increased reactivity indicates that these regions undergo structural changes upon subunit association. 相似文献
7.
Functional analysis of internal transcribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA. 总被引:11,自引:0,他引:11
C A van der Sande M Kwa R W van Nues H van Heerikhuizen H A Raué R J Planta 《Journal of molecular biology》1992,223(4):899-910
Using the previously described "tagged ribosome" (pORCS) system for in vivo mutational analysis of yeast rDNA, we show that small deletions in the 5'-terminal portion of ITS2 completely block maturation of 26 S rRNA at the level of the 29 SB precursor (5.8 S rRNA-ITS2-26 S rRNA). Various deletions in the 3'-terminal part, although severely reducing the efficiency of processing, still allow some mature 26 S rRNA to be formed. On the other hand, none of the ITS2 deletions affect the production of mature 17 S rRNA. Since all of the deletions severely disturb the recently proposed secondary structure of ITS2, these findings suggest an important role for higher order structure of ITS2 in processing. Analysis of the effect of complete or partial replacement of S. cerevisiae ITS2 with its counterpart sequences from Saccharomyces rosei or Hansenula wingei, points to helix V of the secondary structure model as an important element for correct and efficient processing. Direct mutational analysis shows that disruption of base-pairing in the middle of helix V does not detectably affect 26 S rRNA formation. In contrast, introduction of clustered point mutations at the apical end of helix V that both disrupt base-pairing and change the sequence of the loop, severely reduces processing. Since a mutant containing only point mutations in the sequence of the loop produces normal amounts of mature 26 S rRNA, we conclude that the precise (secondary and/or primary) structure at the lower end of helix V, but excluding the loop, is of crucial importance for efficient removal of ITS2. 相似文献
8.
Ribosome synthesis entails the formation of mature rRNAs from long precursor molecules, following a complex pre-rRNA processing pathway. Why the generation of mature rRNA ends is so complicated is unclear. Nor is it understood how pre-rRNA processing is coordinated at distant sites on pre-rRNA molecules. Here we characterized, in budding yeast and human cells, the evolutionarily conserved protein Las1. We found that, in both species, Las1 is required to process ITS2, which separates the 5.8S and 25S/28S rRNAs. In yeast, Las1 is required for pre-rRNA processing at both ends of ITS2. It is required for Rrp6-dependent formation of the 5.8S rRNA 3' end and for Rat1-dependent formation of the 25S rRNA 5' end. We further show that the Rat1-Rai1 5'-3' exoribonuclease (exoRNase) complex functionally connects processing at both ends of the 5.8S rRNA. We suggest that pre-rRNA processing is coordinated at both ends of 5.8S rRNA and both ends of ITS2, which are brought together by pre-rRNA folding, by an RNA processing complex. Consistently, we note the conspicuous presence of ~7- or 8-nucleotide extensions on both ends of 5.8S rRNA precursors and at the 5' end of pre-25S RNAs suggestive of a protected spacer fragment of similar length. 相似文献
9.
Primary and secondary structures of Escherichia coli MRE 600 23S ribosomal RNA. Comparison with models of secondary structure for maize chloroplast 23S rRNA and for large portions of mouse and human 16S mitochondrial rRNAs 总被引:49,自引:34,他引:15
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Christiane Branlant Alain Krol Mohamed Ali Machatt Jean Pouyet Jean-Pierre Ebel Kaylene Edwards Hans Kssel 《Nucleic acids research》1981,9(17):4303-4324
10.
Secondary structure models of the 5.8S rRNA and both internal transcribed spacers (ITS1 and ITS2) are proposed for Calciodinelloideae (Peridiniaceae) and are also plausible for other dinoflagellates. The secondary structure of the 5.8S rRNA corresponds to previously developed models, with two internal paired regions and at least one 5.8S rRNA–28S rRNA interaction. A general secondary structure model of ITS1 for Calciodinelloideae (and other dinoflagellates), consisting of an open multibranch loop with three major helices, is proposed. The homology of these paired regions with those found in other taxa, published in previous studies (e.g. yeast, green algae and Platyhelmithes) remains to be determined. Finally, a general secondary structure model of ITS2 for Calciodinelloideae (and other dinoflagellates) is reconstructed. Based on the 5.8S rRNA–28S rRNA interaction, it consists of a closed multibranch loop, with four major helices. At least helix III and IV have homology with paired regions found in other eukaryotic taxa (e.g. yeast, green algae and vertebrates). Since the secondary structures of both ITS regions are more conserved than the nucleotide sequences, their analysis helps in understanding molecular evolution and increases the number of structural characters. Thus, the structure models developed in this study may be generally useful for future phylogenetic analyses. 相似文献
11.
Characterization of a separate small domain derived from the 5' end of 23S rRNA of an alpha-proteobacterium.
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We demonstrate the presence of a separate processed domain derived from the 5' end of 23S rRNA in ribosomes of Rhodopseudomonas palustris, a member of the alpha-++proteobacteria. Previous sequencing studies predicted intervening sequences (IVS) at homologous positions within the 23S rRNA genes of several alpha-proteobacteria, including R.palustris, and we find a processed 23S rRNA 5' domain in unfractionated RNA from several species. 5.8S rRNA from eukaryotic cytoplasmic large subunit ribosomes and the bacterial processed 23S rRNA 5' domain share homology, possess similar structures and are both derived by processing of large precursors. However, the internal transcribed spacer regions or IVSs separating them from the main large subunit rRNAs are evolutionarily unrelated. Consistent with the difference in sequence, we find that the site and mechanism of IVS processing also differs. Rhodopseudomonas palustris IVS-containing RNA precursors are cleaved in vitro by Escherichia coli RNase III or a similar activity present in R.palustris extracts at a processing site distinct from that found in eukaryotic systems and this results in only partial processing of the IVS. Surprisingly, in a reaction unlike characterized cases of eubacterial IVS processing, an RNA segment larger than the corresponding DNA insertion is removed which contains conserved sequences. These sequences, by analogy, serve to link the 23S rRNA 5' rRNA domains or 5.8S rRNAs to the main portion of other prokaryotic 23S rRNAs or to eukaryotic 28S rRNAs, respectively. 相似文献
12.
The rDNA of eukaryotic organisms is transcribed as the 40S-45S rRNA precursor, and this precursor contains the following segments: 5' - ETS - 18S rRNA - ITS 1 - 5.8S rRNA - ITS 2 - 28S rRNA - 3'. In amphibians, the nucleotide sequences of the rRNA precursor have been completely determined in only two species of Xenopus. In the other amphibian species investigated so far, only the short nucleotide sequences of some rDNA fragments have been reported. We obtained a genomic clone containing the rDNA precursor from the Japanese pond frog Rana nigromaculata and analyzed its nucleotide sequence. The cloned genomic fragment was 4,806 bp long and included the 3'-terminus of 18S rRNA, ITS 1, 5.8S rRNA, ITS 2, and a long portion of 28S rRNA. A comparison of nucleotide sequences among Rana, the two species of Xenopus, and human revealed the following: (1) The 3'-terminus of 18S rRNA and the complete 5.8S rRNA were highly conserved among these four taxa. (2) The regions corresponding to the stem and loop of the secondary structure in 28S rRNA were conserved between Xenopus and Rana, but the rate of substitutions in the loop was higher than that in the stem. Many of the human loop regions had large insertions not seen in amphibians. (3) Two ITS regions had highly diverged sequences that made it difficult to compare the sequences not only between human and frogs, but also between Xenopus and Rana. (4) The short tracts in the ITS regions were strictly conserved between the two Xenopus species, and there was a corresponding sequence for Rana. Our data on the nucleotide sequence of the rRNA precursor from the Japanese pond frog Rana nigromaculata were used to examine the potential usefulness of the rRNA genes and ITS regions for evolutionary studies on frogs, because the rRNA precursor contains both highly conserved regions and rapidly evolving regions. 相似文献
13.
Variable regions V13 and V3 of Saccharomyces cerevisiae contain structural features essential for normal biogenesis and stability of 5.8S and 25S rRNA. 总被引:1,自引:1,他引:0
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R E Jeeninga Y Van Delft M de Graaff-Vincent A Dirks-Mulder J Venema H A Rau 《RNA (New York, N.Y.)》1997,3(5):476-488
The homologous ribosomal RNA species of all organisms can be folded into a common "core" secondary structure. In addition, eukaryotic rRNAs contain a large number of segments, located at fixed positions, that are highly variable in size and sequence from one organism to another. We have investigated the role of the two largest of these variable regions in Saccharomyces cerevisiae 25S rRNA, V13, and V3, by mutational analysis in a yeast strain that can be rendered completely dependent on the synthesis of mutant (pre-)rRNA. We found that approximately half of variable region V13 can be deleted without any phenotypic effect. The remaining portion, however, contains multiple structural features whose disturbance causes serious growth defects or lethality. Accumulation of 25S rRNA is strongly reduced by these mutations, at least in part because they inhibit processing of ITS2. Removal of even a relatively small portion of V3 also strongly reduces the cellular growth rate and larger deletions are lethal. Interestingly, some of the deletions in V3 cause accumulation of 27S(A) pre-rRNA and, moreover, appear to interfere with the close coupling between the processing cleavages at sites A3 and B1(S). These results demonstrate that both variable regions play an important role in 60S subunit formation. 相似文献
14.
15.
Ribosomal RNA of the primitive eukaryote Giardia lamblia: large subunit domain I and potential processing signals 总被引:3,自引:0,他引:3
The cytoplasmic ribosomal RNA (rRNA) from the intestinal protozoan, Giardia lamblia, is unusually short; the large subunit (LS) and small subunit RNA and the 5.8S RNA are only 70-80% of the length found in typical protozoa, and are even smaller than most of their prokaryotic counterparts. Flanking regulatory DNA and processed rRNA sequences are similarly compact in size. To shed light on the origins and implications of this 'minimal' rRNA, the nucleotide sequence encoding the 5.8S RNA and domain I of LS RNA was determined. Secondary structure analysis revealed that an evolutionarily variable internal hairpin is partially 'deleted' in G. lamblia 5.8S RNA; the 3'-terminal pairing with LS RNA is conserved. Previously characterized eukaryotic 'expansion' regions are extensively shortened within the LS RNA; in one case, a hairpin is precisely 'deleted'. The short sequences flanking the mature 5.8S RNA that are removed by RNA processing (ITS1 and ITS2) are C-rich; our analysis suggests that the sequence GCGCCCC, in a hairpin configuration, may function as the processing signal. 相似文献
16.
Assembly of the eukaryotic ribosome requires a large number of trans-acting proteins and small nucleolar RNAs that transiently associate with the precursor rRNA to facilitate its modification, processing and binding with ribosomal proteins. UTPB is a large evolutionarily conserved complex in the 90S small subunit processome that mediates early processing of 18S rRNA. UTPB consists of six proteins Utp1/Pwp1, Utp6, Utp12/Dip2, Utp13, Utp18 and Utp21 and has abundant WD domains. Here, we determined the crystal structure of the tandem WD domain of yeast Utp21 at 2.1 Å resolution, revealing two open-clamshell-shaped β-propellers. The bottom faces of both WD domains harbor several conserved patches that potentially function as molecular binding sites. We show that residues 100–190 of Utp18 bind to the tandem WD domain of Utp21. Structural mapping of previous crosslinking data shows that the WD domains of Utp18 and Utp1 are organized on two opposite sides of the Utp21 WD domains. This study reports the first structure of a UTPB component and provides insight into the structural organization of the UTPB complex. 相似文献
17.
Lieberman KR Firpo MA Herr AJ Nguyenle T Atkins JF Gesteland RF Noller HF 《Journal of molecular biology》2000,297(5):1129-1143
Ribosomal protein L9 consists of two globular alpha/beta domains separated by a nine-turn alpha-helix. We examined the rRNA environment of L9 by chemical footprinting and directed hydroxyl radical probing. We reconstituted L9, or individual domains of L9, with L9-deficient 50 S subunits, or with deproteinized 23 S rRNA. A footprint was identified in domain V of 23 S rRNA that was mainly attributable to N-domain binding. Fe(II) was tethered to L9 via cysteine residues introduced at positions along the alpha-helix and in the C-domain, and derivatized proteins were reconstituted with L9-deficient subunits. Directed hydroxyl radical probing targeted regions of domains I, III, IV, and V of 23 S rRNA, reinforcing the view that 50 S subunit architecture is typified by interwoven rRNA domains. There was a striking correlation between the cleavage patterns from the Fe(II) probes attached to the alpha-helix and their predicted orientations, constraining both the position and orientation of L9, as well as the arrangement of specific elements of 23 S rRNA, in the 50 S subunit. 相似文献
18.
Evolutionarily conserved structural features in the ITS2 of mammalian pre-rRNAs and potential interactions with the snoRNA U8 detected by comparative analysis of new mouse sequences.
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Mechanisms of ITS2 excision from pre-rRNA remain largely elusive. In mammals, at least two endonucleolytic cleavages are involved, which result in the transient accumulation of precursors to 5.8S rRNA termed 8S and 12S RNAs. We have sequenced ITS2 in four new species of the Mus genus and investigated its secondary structure using thermodynamic prediction and comparative approach. Phylogenetic evidence supports an ITS2 folding organized in four domains of secondary structure extending from a preserved structural core. This folding is also largely conserved for the previously available mammalian ITS2 sequences, rat and human, despite their extensive sequence divergence relative to the Mus species. Conserved structural features include the structural core, containing the 3' end of 8S pre-rRNA within a single-stranded sequence, and a stem containing the 3' end of the 12S pre-rRNA species. A putative, phylogenetically preserved pseudoknot has been detected 1 nt downstream from the 12S 3' end. Two long complementarities have also been identified, in sequences conserved among vertebrates, between the pre-rRNA 32S and the snoRNA (small nucleolar RNA) U8 which is required for the excision of Xenopus ITS2. The first complementarity involves the 5.8S-ITS2 junction and 13 nt at the 5' end of U8, whereas the other one occurs between a mature 28S rRNA segment known to be required for ITS2 excision and positions 15-25 of snoRNA U8. These two potential interactions, in combination with ITS2 folding, could organize a functional pocket containing three cleavage sites and key elements for pre-rRNA processing, suggesting a chaperone role for the snoRNA U8. 相似文献
19.
Phylogenetic analysis and predicted secondary structures of the rDNA internal transcribed spacers of the powdery mildew fungi (Erysiphaceae) 总被引:3,自引:0,他引:3
The nucleotide sequences of the internal transcribed spacer (ITS) regions of the ribosomal DNA including the 5.8S rRNA gene
and the 5′ end of the 28S rRNA gene have been determined for 19 species in 10 genera of the powdery mildew fungi in order
to analyze their phylogenetic relationship. These fungi were divided into two large groups based on the nucleotide length
of the ITS regions, and this grouping was in line with that based on the morphological characters of the anamorphic stage
rather than the teleomorphic stage. Although the variable ITS sequences were often ambiguously aligned, conserved sites were
also found. Thus, a neighbor-joining tree was constructed using the nucleotide sequence data of the conserved sites of the
ITS regions, the 5.8S rRNA gene, and the 5′ end of the 28S rRNA gene. The phylogenetic tree displayed the presence of four
groups in the powdery mildews, which were distinguished by their morphology and/or host ranges. In the ITS2 region, the presence
of a common secondary structure having four hairpin domains was suggested, in spite of the highly variable nucleotide sequences
of this region. The predicted secondary structure was supported by the compensatory mutations as well as compensatory conserved
sequences and high G+C content in the predicted stem regions.
Contribution No. 142 from the Laboratory of Plant Pathology, Mie University. 相似文献
20.
Variants within the yeast Ty sequence family encode a class of structurally conserved proteins. 总被引:10,自引:3,他引:7
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A M Fulton J Mellor M J Dobson J Chester J R Warmington K J Indge S G Oliver P de la Paz W Wilson A J Kingsman 《Nucleic acids research》1985,13(11):4097-4112
The Ty transposable elements of Saccharomyces cerevisiae form a heterogeneous family within which two broad structural classes (I and II) exist. The two classes differ by two large substitutions and many restriction sites. We show that, like class I elements a class II element, Tyl-17, also appears to contain at least two major protein coding regions, designated TYA and TYB, and the organisational relationship of these regions has been conserved. The TYA genes of both classes encode proteins, designated p1 proteins, with an approximate molecular weight of 50 Kd and, despite considerable variation between the TYA regions at the DNA level, the structures of these proteins are remarkably similar. These observations strongly suggest that the p1 proteins of Ty elements are functionally significant and that they have been subject to selection. 相似文献