Extensive homologies between the methylated nucleotide sequences in several vertebrate ribosomal ribonucleic acids.

The methylated nucleotide sequences in the rRNA molecules of the following vertebrate cultured cells were compared: human (HeLa); hamster (BHK/C13); mouse (L); chick-embryo fibroblast; Xenopus laevis kidney. In each species the combined 18S, 28S and 5.8S molecules possess approx. 110-115 methyl groups, and the methylated oligonucleotides released after complete digestion of the rRNA by T1 ribonuclease encompass several hundred nucleotides. "Fingerprints" of the three mammalian methyl-labelled 18S rRNA species were qualitatively indistinguishable. "Fingerprints" of digests of 28S rRNA of hamster and mouse L-cells were extremely similar to those of HeLa cells, differing in one and three methylated oligonucleotides respectively. "Fingerprints" of methyl-labelled rRNA from chick and Xenopus strongly resembled those of mammals in most respects, but differed in several oligonucleotides in both 18S and 28S rRNA. At least some of the differences between "fingerprints" appear to be due to single base changes or to the presence or absence of methyl groups at particular points in the primary sequence. The findings strongly suggest that the methylated-nucleotide sequences are at least 95% homologous between the rRNA molecules of the two most distantly related vertebrates compared, man and Xenopus laevis.     

Comparison of nucleotide sequences of large T1 ribonuclease fragments of 18S ribosomal RNA of rat and chicken.

Nucleotide sequences of large T1 ribonuclease fragments of 18S ribosomal RNA of Novikoff rat ascites hepatoma cells and chicken lymphoblastoid cells were determined and compared. Among the 19 large T1 ribonuclease fragments examined of rat 18S ribosomal RNA, 12 fragments were found to be the same in chicken 18S ribosomal RNA. Three fragments of rat 18S ribosomal RNA were not found among large T1 ribonuclease fragments of chicken 18S ribosomal RNA. Four fragments of rat 18S ribosomal RNA were found to be changed in chicken 18S ribosomal RNA. All the changes were point mutations except the change in the largest T1 ribonuclease fragment 1 which is 21 nucleotides long. 2'-0-methylation at the center of the fragment was lost in chicken 18S ribosomal RNA; all the other nucleotides were the same.     

Comparative studies of the primary structures of ribosomal RNAs of several eukaryotic cell lines by the fingerprinting method.

Comparisons of the primary structures of 18S and 28S ribosomal RNAs of man, rat, mouse and chicken were made by two-dimensional fractionation including electrophoresis at pH 3.5 and homochromatography. All large T1 oligonucleotides were recovered from the different fingerprints and their radioactivity was measured. They were then hydrolysed with pancreatic RNase and the pancreatic products were digested with alkali to determine their base composition and detect modified residues. Finally, residues bearing a modification on the ribose were analysed by hydrolyses with snake venom and spleen phosphodiesterases. For the 18A RNAs 23, 27, 26, 24 oligonucleotides, whose lengths range from 22 to 10 residues, were analyzed respectively for man, rat, mouse and chicken. Among these, 14 are identical in the four species, two at least are common to man, rat, mouse but differ by the presence of A-Cps in chicken spot 4' instead of A-Up in spot 4 and A2-Gp in chicken spot 14 instead of A2-Gp in spot 13. For the 28S RNAs of man, rat, mouse and chicken, 20, 19, 21 and 22 oligonucleotides ranging in length from 27 to 12 residues were analyzed. 11 of them are common to the four species; 4 of them are found in man, rat, mouse and one of these (spot 1) has a corresponding spot in chicken from which it differs only by the existence of A3-Up instead of A2-Up. Another mammalian oligonucleotide (spot 6) differs from its homologous chicken spot (spot 6') bytwo point mutations. The same modified residues as found by Khan and Maden in man, chicken, and xenopus, have been found in rat and mouse. Moreover when these modified residues are common to several species they are found within an identical nucleotide sequence, as can be seen in the case of spots 1, 3, 9, 11 of 18S RNAs and 4, 7, 13 for 28S RNAs. The number of differences observed between the ribosomal RNAs of the four species were compared to the number of differences observed in the same species for several proteins, globins alpha and beta, insulin, cytochrome C and lysozyme.     

Identification of the locations of the methyl groups in 18 S ribosomal RNA from Xenopus laevis and man

The 18 S ribosomal RNA from a variety of vertebrate species contains some 40 to 47 methyl groups. The majority of these are 2'-O-ribose substituents; the remaining few are on bases. Several lines of evidence have permitted the identification of the precise locations of the methyl groups in the primary structure of 18 S ribosomal RNA of Xenopus laevis and man. Digestion of RNA with T1 ribonuclease, followed by analysis of the methylated oligonucleotides yielded data on sequences immediately surrounding the methyl groups. Preparative hybridization of X. laevis 18 S ribosomal RNA restriction fragments of ribosomal DNA, followed by fingerprinting analysis on RNA recovered from the hybrids, allowed each methylated oligonucleotide to be mapped to a specific region within 18 S ribosomal RNA. The data on RNA oligonucleotides were correlated with Xenopus ribosomal DNA sequence data in the regions defined by the mapping experiments to identify the precise locations of most of the methyl groups in the X. laevis 18 S RNA sequence. The remaining uncertainties in Xenopus were solved with the aid of data from ribonuclease A fingerprints and, in a few instances, relevant oligonucleotide or sequence data from other laboratories. The locations of most of the methyl groups in human 18 S ribosomal RNA were deduced from the high degree of correspondence between methylated oligonucleotides from human and X. laevis 18 S RNA, together with knowledge of the human 18 S ribosomal DNA sequence. The remaining methylation sites in human 18 S RNA were located with assistance from relevant published comparative data. In the aligned sequences, human and other mammalian 18 S RNA are methylated at all the same positions as in X. laevis, and there are seven additional 2'-O-methylation sites in mammalian 18 S RNA. Further features of the methyl group distribution are briefly reviewed.     

Conservation of the mammalian RNA polymerase II largest-subunit C-terminal domain

Summary We have isolated and sequenced a portion of the gene encoding the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II from three mammals. These mammalian sequences include one rodent and two primate CTDs. Comparisons of the new sequences to mouse and Chinese hamster show a high degree of conservation among the mammalian CTDs. Due to synonymous codon usage, the nucleotide differences between hamster, rat, ape, and human result in no amino acid changes. The amino acid sequence for the mouse CTD appears to have one different amino acid when compared to the other four sequences. Therefore, except for the one variation in mouse, all of the known mammalian CTDs have identical amino acid sequences. This is in marked contrast to the situation among more divergent species. The present study suggests that there is a strong evolutionary pressure to maintain the primary structure of the mammalian CTD. Offprint requests to: J.L. Corden     

Differential distribution of the P1 and P2 protamine gene sequences in eutherian and marsupial mammals and a monotreme

At the protein level, the P1 protamine is the predominant form of mammalian protamine, present in all mammalian spermatozoa analyzed to date. An additional variant, the P2 protamine, has been detected only in spermatozoa of the mouse, hamster and human. Southern blot analysis of a group of restriction enzyme-digested mammalian DNAs has revealed the presence of sequences homologous to the P1 and the P2 mouse protamine genes in diverse species. In agreement with protein studies, nucleotide sequences homologous to the mouse P1 protamine cDNA are widespread, being present in the genomic DNAs of human, rat, dog, ram, horse, bull, hamster, baboon, flying fox (megabat), microbat, boar, North American opossum, and wallaby. Although we detect genomic sequences with strong homology to the mouse protamine 2 cDNA in rat and hamster, we also find weaker but reproducible hybridization to the genomic DNA of human, boar, dog, bull, microbat, wallaby, and platypus. With the exception of the human, the P2 protamine has not been detected in the spermatozoa of these latter species.     

Structural comparison of human, bovine, rat, and Walker 256 carcinosarcoma asparaginyl-tRNA

The complete nucleotide sequences of human placenta, human liver, and bovine liver tRNAAsn have been determined. A comparison of these tRNA structures with the previously reported nucleotide sequences of rat liver and Walker 256 carcinosarcoma tRNAAns reveals that the primary nucleotide sequences of the major species of mammalian cytoplasmic tRNAasn are conserved in higher eucaryotes. The complete nucleotide sequence of these tRNAs is: pG-U-C-U-C-U-G-U-m1G-m2G-C-G-C-A-A-D-C-G-G-D-X-A-G-C-G-C-m2(2)G-psi-psi-C-G-G-C-U-Q(G)-U-U-t6A-A-C-C-G-A-A-A-G-m7G-D-U-G-G-U-G-G-Z-psi-C-G-m1A-G-C-C-C-A-C-C-C-A-G-G-G-A-C-G-C-C-AOH where X is 3-(3-amino-3-carboxyl-n-propyl)uridine, Q is 7-(4,5-cis-dihydroxyl-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosine, Z is an unknown modified nucleotide, and Q(G) represents the replacement of Q nucleoside by G nucleoside in Walker 256 carcinosarcoma tRNAAsn. These primary structures were determined by combined use of the 3H- and 32P-post-labeling techniques. Sequences were compared by tritium nucleoside trialcohol analysis, completed RNAase T1 digestion followed by 3H-labeled fingerprinting on polyethylenimine-impregnated cellulose by two-dimensional thin-layer chromatography (TLC), and polyacrylamide gel electrophoresis of either 5'-32P- and/or 3'-[32P]pCp-labeled tRNA after partial ribonuclease digestions.     

Primary structure of rabbit 18S ribosomal RNA determined by direct RNA sequence analysis

The primary structure of rabbit 18S ribosomal RNA was determined by nucleotide sequence analysis of the RNA directly. The rabbit rRNA was specifically cleaved with T1 ribonuclease, as well as with E. coli RNase H using a Pst 1 DNA linker to generate a specific set of overlapping fragments spanning the entire length of the molecule. Both intact and fragmented 18S rRNA were end-labeled with [32P], base-specifically cleaved enzymatically and chemically and nucleotide sequences determined from long polyacrylamide sequencing gels run in formamide. This approach permitted the detection of both cistron heterogeneities and modified bases. Specific nucleotide sequences within E. coli 16S rRNA previously implicated in polyribosome function, tRNA binding, and subunit association are also conserved within the rabbit 18S rRNA. This conservation suggests the likelihood that these regions have similar functions within the eukaryotic 40S subunit.     

Nucleotide sequence of leucine transfer RNA 1 from Candida (Torulopsis) utilis.

Two species of 32P-labelled leucine tRNA were highly purified from Candida (Torulopsis) utilis by successive column chromatographies. The purified major species of leucine tRNA 1 was completely digested with ribonuclease T1 [EC 3.1.4.8] and with pancreatic ribonuclease A [EC 3.1.4.22]. The resulting fragments were fractionated, and their nucleotide sequences were determined according to Barrell (1). The results of analyses of the two ribonuclease digests were consistent with each other, and indicated that this tRNA is composed of 85 nucleotide residues, including 14 modified nucleotides. A tentative total sequence has been derived on the basis of several features in the cloverleaf structure for tRNA.     

Evolution of nucleic acids coding for ribonucleases: the mRNA sequence of mouse pancreatic ribonuclease

The cDNA of mouse pancreatic mRNA has been cloned. After the library was screened with a rat ribonuclease cDNA probe, the positive clones were isolated and sequenced. There were no differences from the previously determined protein sequence. The mRNA codes for a preribonuclease of 149 amino acid residues including a signal peptide of 25 amino acids. The 3' noncoding region has a length of 260 bp, and the total mRNA length is approximately 940 bp. Comparison with the rat pancreatic ribonuclease sequence showed a high rate of nucleotide substitution. Within the coding region, nonsynonymous and synonymous substitution rates are 4.3 X 10(-9) and 15 X 10(-9) nucleotide substitutions/site/year, respectively. The latter value is one of the highest rates observed in the molecular evolution of mammalian nuclear genes. In the signal sequences the synonymous substitution rate is much lower and about the same as the nonsynonymous rate. Signal sequences of other mouse and rat proteins also exhibit little difference between synonymous and nonsynonymous rates. The sequences of rat and mouse pancreatic ribonuclease messengers were compared with those of bovine pancreatic, seminal, and brain ribonuclease. While the 3' noncoding regions of rat and mouse are very similar, as are those of the three bovine messengers, there is no significant similarity between both rodent and the three bovine messengers for the greater part of these regions. There is a duplication of approximately 50 nucleotides in the 3' noncoding region of the bovine messengers, with a region rich in A and C in between. The presence of this structural feature may be correlated with recent gene duplications that have occurred in the bovine genome.     

The gene encoding hypoxanthine-guanine phosphoribosyltransferase as target for mutational analysis: PCR cloning and sequencing of the cDNA from the rat.

In this paper, the cloning and nucleotide sequence of the cDNA of the rat gene coding for hypoxanthine-guanine phosphoribosyltransferase (hprt) is reported. Knowledge of the cDNA sequence is needed, among other reasons, for the molecular analysis of hprt mutations occurring in rat cells, such as skin fibroblasts isolated according to the granuloma pouch assay. The rat hprt cDNA was synthesized and used as a template for in vitro amplification by PCR. For this purpose, oligonucleotide primers were used, the nucleotide sequences of which were based on mouse and hamster hprt cDNA sequences. Sequence analysis of 1146 bp of the amplified rat hprt cDNA showed a single open reading frame of 654 bp, encoding a protein of 218 amino acids. In the predicted rat hprt amino acid sequence, the proposed functional domains for 5'-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide binding in phosphoribosylating enzymes as well as a region near the carboxyl terminal part were highly conserved when compared with amino acid sequences of other mammalian hprt proteins. Analysis of hprt amino acid sequences of 727 independent hprt mutants from human, mouse, hamster and rat cells bearing single amino acid substitutions revealed that a large variety of amino acid changes were located in these highly conserved regions, suggesting that all 3 domains are important for proper catalytic activity. The suitability of the hprt gene as target for mutational analysis is demonstrated by the fact that amino acid changes in at least 151 of the 218 amino acid residues of the hprt protein result in a 6-thioguanine-resistant phenotype.     

The primary structure of rabbit, calf and bovine liver tRNAPhe.

Highly purified tRNAPhe from rabbit liver, calf liver and bovine liver were completely digested with pancreatic ribonuclease and ribonuclease T1. The oligonucleotides were separated and identified. The tRNAPhe from rabbit liver and calf liver were partially cleaved with ribonuclease T1 or by action of lead acetate. We describe the analyses of the large fragments and the derivation of the primary structure of these mammalian tRNAsPhe.     

Molecular evolution of the mammalian ribosomal protein gene, RPS14

Ribosomal protein S14 genes (RPS14) in eukaryotic species from protozoa to primates exhibit dramatically different intron-exon structures yet share homologous polypeptide-coding sequences. To recognize common features of RPS14 gene architectures in closely related mammalian species and to evaluate similarities in their noncoding DNA sequences, we isolated the intron-containing S14 locus from Chinese hamster ovary (CHO) cell DNA by using a PCR strategy and compared it with human RPS14. We found that rodent and primate S14 genes are composed of identical protein-coding exons interrupted by introns at four conserved DNA sites. However, the structures of corresponding CHO and human RPS14 introns differ significantly. Nonetheless, individual intron splice donor, splice acceptor, and upstream flanking motifs have been conserved within mammalian S14 homologues as well as within RPS14 gene fragments PCR amplified from other vertebrate genera (birds and bony fish). Our data indicate that noncoding, intronic DNA sequences within highly conserved, single-copy ribosomal protein genes are useful molecular landmarks for phylogenetic analysis of closely related vertebrate species.      

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25.

An RNA fragment, constituting three subfragments of nucleotide sequences 1-11, 69-87 and 89-120, is the most ribonuclease-resistant part of the native 5S RNA of Escherichia coli, at 0 degrees C. A smaller fragment of nucleotide sequence 69-87 and 90-110 is ribonuclease-resistant at 25 degrees. Degradation of the L25-5S RNA complex with ribonuclease A or T2 yielded RNA fragments similar to those of the free 5S RNA at 0 degrees C and 25 degrees C; moreover L25 remained strongly bound to both RNA fragments and also produced some opening of the RNA structure in at least two positions. Protein L18 initially protected most of the 5S RNA against ribonuclease digestion, at 0 degrees C, but was then gradually released prior to the formation of the larger RNA fragment. It cannot be concluded, therefore, as it was earlier (Gray et al., 1973), that this RNA fragment contains the primary binding site of L18.     

Structure and organization of the highly repeated and interspersed 1.3 kb EcoRI-Bg1II sequence family in mice.

EcoRI digestion of total mouse DNA yields a prominant 1.3 kb fragment amounting to between 1 and 2% of the mouse genome. The majority of the 1.3 kb EcoRI fragments have a single Bg1II site 800 bp from one end. This EcoRI-Bg1II sequence family shows HindIII and HaeIII sequence heterogeneity. We have cloned representatives of the EcoRI-Bg1II gene family in Charon 16A and studied their structure and organization within the genome. The cloned 1.3 kb fragments show the expected restriction enzyme patterns as well as additional heterogeneity. Representatives of the EcoRI-Bg1II sequence family were found to be interspersed throughout the mouse genome as judged by CsCl density gradient centrifugation experiments. Family members were also found to be organized in higher order repeating units. Homologous sequences were also found in other rodent species including rat and Chinese hamster. Cross hybridization between a cloned 1.3 kb mouse fragment and a cloned CHO repeated sequence is of special interest since the latter has been shown to contain sequences homologous to the Human A1uI family by nucleotide sequencing.     

Studies on the primary structure of the ribosomal 23S RNA of Escherichia coli: II. A characterisation and an alignment of 24 sections spanning the entire molecule and its application to the localisation of specific fragments.

We have employed new methodology to obtain 23S RNA fragments which includes a) the digestion of the RNA within 50S subunits and b) the limited hydrolysis of the 13S and 18S fragments. By comparing all 23S RNA fragments, obtained heretofore, we have characterised and aligned 24 sections of this RNA spanning nearly the entire molecule. These results allow the localisation of any new 23S RNA fragment by comparison of the fingerprint of its T1 ribonuclease digest to the characteristic ones of the different sections. In this way we obtained a more definite localisation of the binding sites of the 50S proteins L1, L5, L9, L18, L20, L23 and L25. We also specified a ribonuclease sensitive region of 23S RNA in native 50S subunits, extending from the 1100th nucleotide from the 5' end to the 1000th nucleotide from the 3' end; this region contains a cluster of 5 modified nucleotides and may be at the subunit interface.     

The nucleotide sequence of cysteine transfer ribonucleic acid from baker''s yeast. Identification of the products from partial degradation of the molecule and derivation of the complete sequence.

1. A series of large oligonucleotide fragments derived from tRNA Cys, were separated chromatographically and the sequence of each was deduced by examination of the products of digestion with pancreatic and T1 ribonucleases. 2. The location of the specific cleavage points in the nucleotide chain was similar to that produced by brief treatment with pancreatic ribonuclease. 3. The fragments could be arranged into two alternative sequences. The correct sequence was deduced by the sequential removal and identification of the first nine nucleotides from the 3'-end of the terminal half of the molecules.