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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.

Animals

Nuclear retention of 18S ribosomal RNA by human myeloma cells.

Normal quiescent lymphocytes regulate their ribosome content by selectively degrading newly synthesized 18S ribosomal RNA. Unlike actively dividing HeLa cells, lymphocytes retain 18S ribosomal RNA in the nucleus after synthesis instead of immediately transporting it to the cytoplasm. Subcellular fractionation of the highly differentiated human neoplastic lymphocyte RPMI-8226 reveals that this cell line also retains 18S ribosomal RNA in the nucleus, a trait not displayed by the less differentiated human lymphoblastoid cell line RPMI-4265. These observations suggest that neoplastic cells can be phenotypically characterized by their ribosomal RNA processing patterns.

Animals

Isolation from rat liver and sequence of a RNA fragment containing 32 nucleotides from position 5 to 36 from the 3' end of ribosomal 18S RNA.

Crude tRNA isolated from rat liver by the method of Rogg et al. (Biochem. Biophys. Acta 195, 13-15 1969) contains N6-dimethyladenosine (m6-2A) and was therefore fractionated in order to identify the m6-2A-containing RNAs. A unique species of RNA was purified which contained all the m62A present in the crude tRNA. Sequence analysis by postlabeling with gamma-32p-ATP and polynucleotide kinase revealed that this RNA represents the 32 nucleotides AAGGUUUC(C)U GUAGGUGm62Am62ACCUGCGGAAGGAUC from position 5 to 36 of the 3' terminus of ribosomal 18S RNA. The 36 nucleotide long sequence from the 3' end of rat liver 18S rRNA exhibits extensive homology with the corresponding sequence of E. coli 16S rRNA and with the 21 nucleotide long 3' terminal sequence so far known from Saccharomyces carlsbergensis 17S rRNA. A heterogeneity in this sequence provides the first evidence on the molecular level for the existence of (at least) two sets of redundant ribosomal 18S RNA genes in the rat.

Adenine Nucleotides

Wheat embryo mitochondrial 18S ribosomal RNA: evidence for its prokaryotic nature.

We present a catalog of sequences of oligonucleotides produced by T1 ribonuclease digestion of 32P-labeled small-ribosomal-subunit RNA ("18S rRNA) isolated from purified wheat embryo mitochondria. This catalog is compared to catalogs published for prokaryotic and chloroplast 16S rRNAs and to preliminary results for wheat cytosol 18S rRNA. These comparisons indicate that: (1) wheat mitochondrial 18S rRNA is clearly prokaryotic in nature, showing significantly more sequence homology with 16S rRNAs than can be expected to arise by chance (p less than 0.000001); (2) shared oligonucleotide sequences include an especially high proportion of those identified as conserved in the evolution of prokaryotic rRNAs; and (3) wheat embryo mitochondrial and cytosol 18S rRNAs retain no more, and perhaps less, than the minimum sequence homology detectable by this sensitive method. These results argue in favor of an endosymbiotic origin for mitochondria.

Base Sequence

Evolutionary trends in 18S ribosomal RNA nucleotide sequences of rat, mouse, hamster and man.

The large T1 ribonuclease fragments of 18S ribosomal RNA from four mammalian species, rat, mouse, hamster and man, were compared by two-dimensional homochromatography fingerprinting. The nucleotide sequences of the large T1 ribonuclease fragments, polypyrimidines and polypurines which were different among the four mammalian species were determined and compared. The method used for determining nucleotide sequences utilizes 32p-labeling of oligonucleotides at their 5'-termini by polynucleotide kinase, partial digestion by ribonucleases and analysis of labeled spots by homochromatography-fingerprinting. Several examples of point mutations were detected. It was of interest that the 18S rRNA of Chinese hamster has more oligonucleotide sequences in common with those of man that rat or mouse.

Animals

Sequence analysis of T1 ribonuclease fragments of 18S ribosomal RNA by 5'-terminal labeling, partial digestion, and homochromatography fingerprinting.

The method employed to determine the sequence of a T1 RNase fragment, A-A-A-A-A-U-A-A-C-A-A-U-A-C-A-Gp, from Novikoff rat hepatoma 18S ribosomal RNA is described. This method is applicable to any oligoribonucleotide produced by specific endonucleases that leave the newly cleaved 5'-end free for labeling with polynucleotide kinase and gamma-(32p)-ATP. The (32p)-labeled oligoribonucleotide is subjected to partial endonucleolytic digestion and fractionated by two-dimensional homochromatography fingerprinting. The nucleotide sequence is determined by following mobility shifts of the labeled and partially digested oligoribonucleotides in homochromatography fingerprinting.

Animals

Modified nucleotides in T1 RNase oligonucleotides of 18S ribosomal RNA of the Novikoff hepatoma.

The primary structure of 18S rRNA of the Novikoff hepatoma cells was investigated. Regardless of whether the primary sequence of 18S rRNA is finally determined by RNA sequencing methods or DNA sequencing methods, it is important to identify numbers and types of the modified nucleotides and accordingly the present study was designed to localize the modified regions in T1 RNase derived oligonucleotide. Modified nucleotides found in 66 different oligonucleotide sequences included 2 m62A, 1 m6A, 1 m7G, 1m1cap3psi, 7 Cm, 13 Am, 9 Gm, 11 Um, and 38 psi residues. A number of these modified nucleotides are now placed in defined sequences of T1 RNase oligonucleotides which are now being searched for in larger fragments derived from partial T1 RNase digests of 18S rRNA. Improved homochromatography fingerprinting (Choi et al. (1976) Cancer Res. 36, 4301) of T1 RNase derived oligonucleotides provided a distinctive pattern for 18S rRNA of Novikoff hepatoma ascites cells. The 116 spots obtained by homochromatography contain 176 oligonucleotide sequences.

Base Sequence

Sequence determination of the 3' terminal T1 oligonucleotide of 18S ribosomal RNA.

We have reexamined the primary structure of the 3' terminal oligonucleotide of 18S RNA from chicken fibroblasts and have shown, contrary to previously published results that this extremity G-A-U-C-A-U-U-AOH is identical to that of the rabbit, drosophila and bombyx. Furthermore the electrophoretic mobility and composition of the 3' terminal oligonucleotides of 18S RNA from rat and human cells are similar to that of other RNAs and show that the identity of structure for this region of 18S RNA extends to include all tested species between yeast and man. This finding reveals a marked degree of evolutionary constraint on the structure of this region.

Animals

Hypermodified alkali-stable dinucleotide sequences in each of the high-molecular-weight (26S and 18S) ribosomal RNA species of wheat.

Two hypermodified, alkali-stable dinucleotide sequences, each containing a base modification in addition to sugar methylation, are known to be present in wheat embryo 26S + 18S rRNA (Gray, M.W. (1974) Biochemistry 13, 5453-5463). Quantitative analysis of unfractionated 26S + 18S rRNA had suggested that each of these sequences (Cm-psi p and psi m-Ap, where Cm=O2'-methylcytidine and psi m-O2'-methylpseudouridine) was present in either the 18S or the 26S rRNA species, but not the both, at a frequency of not more than once per chain. In the study reported here, the individual 32P-labeled 18S and 26S rRNA species were isolated from viable wheat embryos germinated in the presence of [32P]orthophosphate. From analyses of phosphodiesterase and alkaline hydrolysates of the separated [32P]RNAs, we conclude that psi m-Ap is confined to wheat cytosol 18S rRNA, whereas Cm-psi p is localized in wheat cytosol 26S rRNA. The presence of psi m in the 18S rRNA of wheat stands in contrast with the situation in animal cells, where this hypermodified nucleoside is located in the 28S rRNA (Khan, M.S.N. & Maden, B.E.H. (1976) J. Mol. Biol. 101, 235-254).

Cytosol

Chromosome location of the ribosomal genes in Triturus vulgaris meridionalis (Amphibia Urodela). III. Inheritance of the chromosomal sites for 18S + 28S ribosomal RNA.

In Triturus vulgaris meridionalis, the 18S + 28S rDNA sequences have been shown to be located in a number of additional chromosomal sites besides the nucleolus organizing region. The additional ribosomal sites have been found to vary as to their number and chromosomal location in different individuals of the species.--The data presented in this study concern the chromosomal distribution of the ribosomal sequences as analyzed by in situ hybridization technique in two individuals as well as in their offspring. The evidence obtained by this analysis indicates quite clearly that all 18S + 28S rRNA sites present in each individual genome are inherited according to simple mendelian principles.

Animals

Identification of the 5S RNA binding site in intermolecular complexes of wheat embryo ribosomal 5S and 18S RNA.

The nucleotide sequence in wheat embryo ribosomal 5S RNA that binds by complementary base pairing to ribosomal 18S RNA has been identified. The 5S--18S RNA complex was formed in vitro and the interacting regions of the two RNA chains were isolated by a combination of nuclease digestion and electrophoresis on polyacrylamide gels. The binding site is close to the 3'-end of the 5S RNA molecule and contains a maximum of 24 nucleotides.

Base Sequence

Chromosome location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia, Urodela). II. Intraspecific variability in number and position of the chromosome loci for 18S + 28S ribosomal RNA.

Ribosomal genes have been localized on mitotic and lampbrush chromosomes of 20 specimens of Triturus vulgaris meridionalis by in situ hybridization with 3H 18S + 28S rRNA. The results may be summarized as follows: 1) each individual shows positive in situ hybridization at the nucleolus organizing region (NOR) on chromosome XI; 2) in addition, many specimens exhibit a positive reaction in chromosomal sites other than the NOR (additional ribosomal sites); 3) the chromosomal distribution of the additional sites appears to be identical in different tissues from the same specimen and to follow a specific individual pattern; 4) the additional ribosomal sites are preferentially found at the telomeric, centromeric or C-band regions of the chromosomes involved.

Animals

Characterization of 5.8S ribosomal ribonucleic acid in Neurospora crassa.

Neurospora crassa ribosomes contain a species of ribonucleic acid (RNA) of molecular weight 54,000, similar to 5.8S ribosomal RNA previously described for other eukaryotic organisms. The 5.8S RNA from N. crassa was found to be released by heat treatment at 60 C from 25S ribosomal RNA but not from 18S ribosomal RNA. The base composition of N. crassa 5.8S RNA was similar to that of 5.8S RNA from Saccharomyces cerevisiae, but differed from animal 5.8S RNA. During the course of this study, it was discovered that N. crassa 25S ribosomal RNA had a number of internal cleavages that may exist in vivo.

Cytidine

The synthesis of 5S RNA and its relationship to 18S and 28S ribosomal RNA in the bobbed mutants of Drosophila melanogaster.

Ribosomes contain one molecule each of 5S, 18S and 28S RNA. In Drosophila melanogaster although the genes for 18S + 28S are physically separated from the 5S RNA genes, the multiplicity of various ribosomal RNA genes is roughly the same. Thus a coordinate synthesis of these three molecules might seem feasible. This problem has been approached by determining the molar ratios of various RNA's in ovaries and in adult flies. In ovaries there is a slight excess of 5S RNA molecules over other rRNA's, but in adult flies no such differences exist. Bobbed mutants also have the same molar ratios as wild-type flies. Results on 5S RNA synthesis in both in vitro and in vivo studies show that it is reduced in coordination with 18S + 28S rRNA in the bobbed mutants of Drosophila melanogaster. Various possibilities are discussed in considering the implications of these results.

DNA

Base sequence complexity of the stable RNA species of Drosophila melanogaster.

The base sequence complexity of Drosophila transfer RNA (tRNA), 5S RNA, and 18S + 28S ribosomal RNA was determined by analyzing the kinetics of RNA-DNA hybridization on membrane filters. We find that Drosophila tRNA is made up from about 59 basic nucleotide sequences distinguishable by hybridization, suggesting that many of the 99 tRNA species resolved by reverse phase chromatography (RPC-5) are homogenic. In contrast 5S RNA was found to contain a single family of sequences. Either 18S ribosomal RNA (rRNA) alone, or 18S + 28S rRNA together, behaved kinetically as two sequence families, and the possible basis for this unexpected result is discussed.

Animals

Cytological localization of the genes for the four classes of ribosomal RNA (25S, 18S, 5.8S and 5S) in polytene chromosomes of Phaseolus coccineus.

Homologous tritiated 25S, 18S and 5.8S rRNAs were used separately for in situ hybridization to the polytene chromosomes of the embryo suspensor cells of phaseolus coccineus. Hybridization occurred at the same chromosomal sites which were labeled in previous in situ hybridization experiments with 25 + 18S rRNAs in the same material (Avanzi et al., 1972), namely: nucleolus organizing system (satellite, nucleolar constriction and organizer) of chromosome pairs I (S1) and V (S2), proximal heterochromatic segment of the long arm of chromosome pair I, and terminal heterochromatic segment of chromosome pair II. Competition hybridization experiments confirmed for P. coccineus the high sequence homology between 25S and 18S rRNA already known for other plants. Homologous 125I-5S rRNA was found to hybridize to three sites in the polytene chromosomes of P. coccineus: the proximal heterochromatic segment in the long arm of chromosome pair I (which also bears the sequences complementary to 25S, 18S and 5.8S RNAs), most of the proximal heterochromatic segment plus a small portion of adjoining euchromatin in the long arm of chromosome pair VI and the large intercalary heterochromatic segment in the same chromosome pair. Simultaneous labeling of the two 5S RNA sites in chromosome VI was quite rare (3%), the rule being labelling of one site to the exclusion of the other, with a labeling frequency of 43.7% and 53.3% for sites no. 1 and no. 2 respectively. These results are interpreted as being due to differential hybridizability of chromosomal sites such as described in other materials.

Chromosome Mapping

Variability of bacterial gene-directed enzyme production in human genetically deficient cells.

Human beta-galactosidase-deficient skin fibroblasts from a patient with generalized gangliosidosis (GMI-gangliosidosis type I) were treated with phage lambda plac DNA, coding for Escherichia coli beta-galactosidase (beta-D-galactoside galactohydrolase, EC.3.2.1.23). New beta-galactosidase activity detected in cell extracts of phage DNA-treated GMI-gangliosidosis fibroblasts continued to vary considerably from one experiment to another. It behaved like the E. coli z-gene product upon immunochemical and physicochemical investigation. In some experiments the antigenic behavior of resultant beta-galactoside activity in lambda plac DNA-treated cells resembled that of mutant E. coli beta-galactosidase. Among the factors and variables that may be responsible for the variation in the results obtained here and elsewhere, low physical binding between prokaryotic mRNA sequences and fibroblast ribosomal RNA could play a part connected with effective translation. This hypothesis is discussed under the aspect of a comparison of the ribosomal binding site of lac z mRNA with the 3'-terminus of the eukaryotic 18s ribosomal RNA, which shows limited possibilities for base-pairing interactions. More extensive possibilities for forming Watson-Crick base pairs between their initiation site and the eukaryotic ribosomal binding site exist for other prokaryotic messengers, such as those of Q beta-replicase, f 1-coat protein, or UDPG-4-epimerase.

Cells, Cultured