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Growing and confluent cultures of mouse fibroblasts were labeled with 3H-uridine and chased with an excess of nonradioactive uridine to investigate the turnover of ribosomal RNA. Growing cultures did not turn over their 18S and 28S ribosomal RNA; however, confluent cultures did show ribosomal RNA (rRNA) turnover. If the cells were labeled while growing, and chased when confluent, 18S RNA displayed a two-component decay curve, while 28S RNA showed only single-component decay, similar in lifetime to the first component of the 18S RNA decay curve. If the cells were labeled while confluent, both the 18S and 28S RNA showed single-component decay curves with a lifetime possibly only slightly longer than the lifetime of the first component of the 18S RNA and the single component of the 28S RNA of the cultures labeled while growing.

The nucleotide sequence of the 5S ribosomal RNA of somatic cells of the chicken (Gallus gallus) differs from that of other vertebrates thus far examined. Besides base substitutions, alterations include two nucleotide deletions and two additions.

Ribosomal proteins extracted from chloroplasts of wild-type and a streptomycin-resistant mutant of Euglena gracilis were analyzed by two-dimensional gel electrophoresis. Comparison between electrophoretograms obtained from both strains indicates that 4 acidic proteins found in the 30 S ribosomal subunit of wild-type are missing from the 30 S ribosomal subunit of the streptomycin-resistant mutant. These proteins might be part of the binding site of the antibiotic.

Ribosomal RNA and precursor ribosomal RNA from at least one representative of each vertebrate class have been analyzed by electron microscopic secondary structure mapping. Reproducible patterns of hairpin loops were found in both 28 S ribosomal and precursor ribosomal RNA, whereas almost all the 18 S ribosomal RNA molecules lack secondary structure under the spreading conditions used. The precursor ribosomal RNA of all species analyzed have a common design. The 28 S ribosomal RNA is located at or near the presumed 5′-end and is separated from the 18 S ribosomal RNA region by the internal spacer region. In addition there is an external spacer region at the 3′-end of all precursor ribosomal RNA molecules. Changes in the length of these spacer regions are mainly responsible for the increase in size of the precursor ribosomal RNA during vertebrate evolution. In cold blooded vertebrates the precursor contains two short spacer regions; in birds the precursor bears a long internal and a short external spacer region, and in mammals it has two long spacer regions. The molecular weights, as determined from the electron micrographs, are 2·6 to 2·8 × 10 6 for the precursor ribosomal RNA of cold blooded vertebrates, 3·7 to 3·9 × 10 6 for the precursor of birds, and 4·2 to 4·7 × 10 6 for the mammalian precursor. Ribosomal RNA and precursor ribosomal RNA of mammals have a higher proportion of secondary structure loops when compared to lower vertebrates. This observation was confirmed by digesting ribosomal RNAs and precursor ribosomal RNAs with single-strandspecific S 1 nuclease in aqueous solution. Analysis of the double-stranded, S 1 -resistant fragments indicates that there is a direct relationship between the hairpin loops seen in the electron microscope and secondary structure in aqueous solution.

Transcription of ribosomal protein genes in the str-spc region in Escherichia coli seems to be exclusively anticlockwise. Insertion mutations in this region inactivate many but not all distal ribosomal protein genes. Thus, there seems to be more than one transcriptional unit for the ribosomal protein genes in this region.

A procedure for the quantitative determination of one RNA minor base in approximately 1800–2000 nucleotides is described. This procedure is useful for the analysis of minor base content in systems where in vivo labeling of RNAs to high specific activity is not readily achieved.

Summary When incubated in vitro with EDTA, ribosomal RNA in fetal rat liver polysomes was degraded to produce fragments some of which have molecular weights similar to those of in vivo degradation intermediates of 28S ribosomal RNA. The degradation occurred endonucleolytically and was inhibited by SDS and liver “cell sap”.

Summary Large T 1 RNase fragments of 18S and 28S rRNA and their nucleolar precursors were separated by successive elution from columns of DEAE Sephadex at pH 7.6 and DEAE-cellulose at pH 3.4, respectively. The fragments that contain the methylated oligonucleotides Am-Gm-Cm-Ap, Um-Gm-Up, Gm-Up and Cm-Ap are referred to as α2 −2 , α2 −4 , α2 −3 and α1 −1 ′. Fragment α2 −2 is the eicosanucleotide Am-Gm-Cm-A-A-A-U-U-C-A-U-A-U-U-C-A-A-A-C-Gp which is the 5′ end of 28S rRNA and its precursors including nucleolar 45S RNA. The dinucleotide Cm-Ap is present only in 18S rRNA and 45S nucleolar RNA.

Several earlier autoradiographic observations have reported the absence or the very low rate of nuclear incorporation of RNA precursors in male gametocytes. The problem of ribosomal RNA synthesis in mouse spermatocytes was approached biochemically by using ionizing radiation which at low doses selectively destroys spermatogonia without affecting the other germ cells and Sertoli cells. With this method two testicular populations were obtained, one composed by spermatocytes, spermatids and Sertoli cells (group B) and the other only by Sertoli cells and late spermatids (group C). The control and irradiated animals were injected intraperitoneally with 32 PO 4 and at various intervals thereafter the RNA was extracted from the seminiferous tubules and analysed by sucrose gradient sedimentation. It was possible to rule out effects of radiation on the rate of incorporation or of breakdown of radioactive RNA within the dose range used. Since spermatids are inactive in RNA synthesis the difference in the rate of incorporation into rRNA between the control and group B animals and between group B and group C animals should indicate the activity of spermatogonia and spermatocytes, respectively. The results indicate that the radioactivity incorporated into 18 and 28S ribosomal RNA is practically the same in group B and group C testes 3 and 6 h after labelling, but increases at a faster rate in the former than in the latter at long intervals after 32 PO 4 administration. Three hypotheses are discussed to explain these results. One is that during meiosis there is a deceleration of rRNA transcription so that long periods of labelling are needed to detect the activity of the spermatocytes. This hypothesis seems unlikely on the basis of actinomycin D experiments. Another interpretation is that the rate of maturation of ribosomal precursors is greatly reduced during male meiosis, thus leading to an accumulation of ribosomal precursors in the nucleolus and to a delayed appearance in the cytoplasm of mature ribosomal molecules. Finally, a third hypothesis is that spermatocytes do not synthesize appreciable amounts of rRNA during meiotic prophase and that there is a transfer of rRNA from the Sertoli cell into spermatocytes.

Eco R 1 restriction endonuclease makes two cuts in each repeating unit of amplified ribosomal DNA (rDNA) from Xenopus laevis . The locations of these cuts have been established by comparison of the secondary structure of single-stranded Eco R 1 fragments (as visualized in the electron microscope) with that of X. laevis rRNAs and of long strands of uncut rDNA. Of the two classes of fragments generated, the smaller one contains 90% of the 28S rRNA gene, has a duplex molecular weight of 3.0 × 10 6 and is homogeneous in size. The larger class of molecules contains 80% of the 18S rRNA gene and all of the nontranscribed spacer. These latter fragments are heterogeneous with molecular weights ranging from 4.0 to 5.9 × 10 6 . The distribution of sizes within the large class of fragments varies among different preparations of rDNA, and heterogeneity is present in the DNA amplified from the rDNA of a single nucleolar organizer. The heterogeneity is located in the nontranscribed spacer region which is variable in length. This has been demonstrated by the formation of deletion loops in heteroduplexes made between larger fragments of different lengths.

A method for preparation of highly active mammalian ribosomal subunits is described, which yields 60S subunits containing no more than 33% protein. It is suggested that the composition of these subunits corresponds closely to that of Escherichia coli 50S subunits. Data on the composition of bacterial and mammalian ribosomal subunits recovered from CsCl are given. It is shown that the commonly employed assumptions about the relationship between the buoyant density of a particle in CsCl and its protein content are in error. The composition of ribosomal subunits cannot ordinarily be calculated from the buoyant density in CsCl.

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.

Oligonucleotides, produced by digestion of 32P-labeled 18 S ribosomal RNAs from Novikoff rat ascites hepatoma and HeLa cells with T1 ribonuclease, were mapped by homochromatography fingerprinting. The fingerprint patterns from the two rRNAs were quite similar and 18 of the separated large fragments had corresponding migrations but one fragment present in the rat 18 S rRNA was absent from the human 18 S rRNA. The nucleotide sequence assigned to this fragment is A-C-C-C-C-C-C-U-U-C-C-C-Gp.

The molecular weights of Escherichia coli 30 S pre-ribosomal RNA, and of the 25 S and 17·5 S products of cleavage by RNAase III, have been determined by formamide gel electrophoresis and electron microscopy. They are 2·10, 1·20, and 0·65 × 10 6 ± 2·5%. Thus, the 30 S pre-ribosomal RNA contains about 22% of sequences in addition to 16 S and 23 S ribosomal RNA. Combining these results with others in the literature, a more detailed processing pathway for E. coli ribosomal RNA can be deduced.

The 3′ hydroxyl end of 28S L-rRNA (major RNA species of the larger subribosomal particle) was labelled by coupling its 2-hydroxy-3-naphthoic acid hydrazine with diazotized [3H]aniline. The RNA was hydrolysed partially with ribonuclease T1 and fractionated on Sephadex G-200. The results show that a highly structured segment with 78% G+C content and a number-average molecular weight of at least 1.0×10(5)-1.8×10(5) is located at the 3′ hydroxyl end of the 28S rRNA molecule.

In situ hybridization with 3H 18S and 28S ribosomal RNA from Xenopus laevis has been used to study the distribution of DNA sequences coding for these RNAs (the nucleolus organizing regions) in the genomes of six mammals. Several patterns of distribution have been found: 1) A single major site (rat kangaroo, Seba's fruit bat), 2) Two major sites (Indian muntjac), 3) Multiple sites in centromeric heterochromatin (field vole), 4) Multiple sites in heterochromatic short arms (Peromyscus eremicus), 5) Multiple sites in telomeric regions (Chinese hamster). - The chromosomal sites which bind 3H 18S and 28S ribosomal RNA correspond closely to the sites of secondary constrictions where these are known. However, the correlation is not absolute. Some secondary constrictions do not appear to bind 3H ribosomal RNA. Some regions which bind ribosomal RNA do not appear as secondary constrictions in metaphase chromosomes. - Although the nucleolus organizing regions of most mammalian karyotypes are found on the autosomes, the X chromosomes in Carollia perspicillata and C. castanea carry large clusters of sequences complementary to ribosomal RNA. In situ hybridization shows that the Y chromosome in C. castanea also has a large nucleolus organizing region.

Transcriptional units for ribosomal proteins in Escherichia coli were measured using the ultraviolet sensitivities of the rates of synthesis of individual ribosomal proteins. The ultraviolet sensitivities of gene transcriptions are proportional to the distances from the promoters. The longest transcriptional units for ribosomal proteins are 3.6 x 10(6) of DNA molecular weight corresponding to 1.8 x 10(6) of RNA or to 180 000 of protein. The length would cover 10--12 genes of ribosomal proteins (of an average Mr of 15000-18000).

Only two sites in 5 S RNA react with Kethoxal in 50 S ribosomal subunits. These two sites, G 13 and G 41 , have previously been found to be accessible in free 5 S RNA. Nucleotide sequences which have been suggested as possible binding sites for the T-ψ-C-G loop of tRNA are not accessible.

Although individual ribosomal proteins are synthesized at the same rate during exponential growth, during a nutritional shift-up, they are synthesized at different rates. Proteins S6, S10, S14, S20 and L2, L3, L4 and L13 are synthesized to the greatest extent. Proteins S2, S3, S4, S13, S17 and L7, L8, L9, L25, L29 and L30 are synthesized at the lowest rate. Although the average rate of synthesis of 50-S protein is greater than that for 30-S protein during a shift-up, the data on rates of synthesis of individual proteins do not support such a distinction for each individual ribosomal protein. The extent of increased synthesis for the individual ribosomal proteins of the 30-S subunits correlated well with the assembly order of these proteins. This correlation is not as striking for the 50-S subunit.

High-molecular-weight yeast ribosomal DNA, purified by preparative Hg2/Cs2SO4 gradient centrifugation, was characterized using RNA-DNA hybridization, CsCl gradient analysis and denaturation-renaturation experiments. The following conclusions are drawn from the results of these studies. 1 55–60% of the total yeast rDNA is made up of sequences coding for 42-S ribosomal precursor RNA. 2 The mean length of the 42-S precursor cistrons corresponds to a molecular weight of 6.2 × 106. 3 The overall G + C content of yeast rDNA is 42%. On the other hand, both the segments coding for the nonribosomal excess RNA of the 42-S precursor and those specifying 26-S and 17-S rRNA have a higher G + C content of 46.9–47.5%. 4 The 42-S precursor cistrons are closely bound up with sequences rather low in G + C content (34–35%), which account for 40–45% of the total yeast rDNA. In contrast to the multiple 42-S precursor cistrons which show very little or no base sequence divergence these intervening (A + T)-rich sequences display a considerable heterogeneity in base sequence.

Specialized lambda transducing phages have been isolated carrying approximately half the ribosomal protein genes of E. coli. These phages carry regions of the bacterial chromosome between aroE and fus. The ribosomal protein genes on these phages have been identified by the stimulation of ribosomal protein synthesis in ultraviolet-irradiated bacteria following infection by the transducing phage, and by the in vitro synthesis of ribosomal proteins in a DNA-dependent protein synthesizing system. The results indicate lambdadspcl probably carries at least 22 ribosomal protein genes and lambdadspc2 at least 26 genes. All these genes are clustered between trkA and strA. At least 13 of them have not been previously mapped.

The sequence of Chlorella cytoplasmic 5 S RNA has been determined by fingerprinting techniques. Partial digests were fractionated by a two-dimensional acrylamide gel electrophoretic technique, which indicates whether specific fragments are paired in the molecule. In this way, the four main base-paired regions of the molecule were located. The sequence of Chlorella cytoplasmic 5 S RNA is related to, but different from, that of other eukaryotic 5 S RNAs: it shows approximately 60% homology with vertebrate 5 S RNA and 40% homology with yeast 5 S RNA. In some respects the conformation of the molecule in solution is quite different from that of other sequenced 5 S RNAs: in particular, the highly accessible region found around position 40 in all other 5 S RNAs (prokaryotic and eukaryotic) does not exist in this molecule.

Centrifugation of DNA from Euglena gracilis in Hg2+-CS2SO4 equilibrium gradients allows the chloroplast DNA to be separated clearly from the nuclear DNA. Cytoplasmic ribosomal RNA isolated from a heat-bleached strain hybridizes only to the nuclear DNA. The ribosomal RNA cistrons in the chloroplast DNA are not related to those in the nuclear DNA. A possible origin for the chloroplast genome is suggested by the observation that roughly one-fourth of the ribosomal RNA complementary sequences in chloroplast DNA anneal with RNA from the blue-green alga Anacystis nidulans.

The kinetics of the rate of synthesis of the ribosomal proteins has been determined during a nutritional shift-up in E. coli. Evidence is obtained for a maximal transcription time of 2 min for possible transcriptional units for ribosomal proteins. This is in accordance with the earlier conclusion that ribosomal protein genes are clustered in several relatively small transcriptional units (Molin et al., 1974).

Previtellogenic oocytes of Xenopus laevis synthesize large amounts of 5 S RNA and transfer RNA, but very little, if any, 28 S and 18 S RNA. About half of the RNA of these oocytes is stored in nucleoprotein particles sedimenting at 42 S. These particles contain 5 S RNA, transfer RNA, and several proteins, the function of which remains so far unknown. The proteins of the 42 S particles were analyzed by two-dimensional electrophoresis on polyacrylamide gel. The resulting fingerprints displayed one major and two minor basic spots. None of these coincided with any of the 37 spots produced by the 60 S subunit of the ribosomes and with the 30 spots produced by the 40 S subunit. We conclude that no ribosomal component other than 5 S RNA is present in the 42 S particles. The fingerprints of 40 S and 60 S ribosomal proteins from X. laevis coincided almost completely with the corresponding fingerprints from the rat and the rabbit.

The MS2 RNA binding capacity of 30S ribosomal subunits, which is lost when protein S1 is removed, can be restored following incubation with interference factor iα and repelleting. Polyacrylamide-agarose gel electrophoresis shows that, under these conditions, a faster moving, non-RNA binding 30S species, which contains no S1, is converted to a slower moving RNA-binding one, having the same mobility as the 30S species that contains protein S1. Factor iα binds to single-stranded RNAs in a pattern that closely resembles the RNA binding pattern of initiation factor IF-3.

If RNA is extracted from the ribosomes which had been isolated from frozen-thawed tissue of Galleria mellonella, the 28 S RNA, when heated or treated with urea, dissociates into seven different species of polynucleotide fragments. They were designated as R1, R2, R3, R4, R5, R6, and R7, whose molecular weights were estimated to be 1.15x10-6, 0.75x10-6, 0.55x10-6, 0.40x10-6, 0.30x10-6, 0.25x10-6, 0.20x10-6 daltons, respectively. It is likely that R1 and R5 arise from a single nick in original 38 S rRNA. Experiments with isolated R1 suggest that it is made up of a hydrogen-bonded complex of R2 and R4. R5 is a complex of R6 and an unidentified species, X. It is suggested that these fragments result from nicks which are introduced, secondarily, in the phosphodiester bonds by an endogenous endonuclease(s). Since the secondary nicks are limited in number and located in specific points of the molecule, it appears that the reaction is quite specific. It was also shown that the 28 S aphid RNA, which apparently lacks the primary nick, is susceptible to nicking.

Nucleoli isolated from the Novikoff rat tumor were used to study the biosynthesis and methylation of ribosomal RNA in vitro . Nucleolar nascent preribosomal RNA was labeled in limited regions with methyl - 3 H groups by incubation of nucleoli with S -adenosyl- l -[ methyl - 3 H]methionine under conditions which minimize degradation and further elongation of the RNA chains. The labeled nucleolar RNA was subsequently extracted by phenol treatment and fractionated by sucrose gradient centrifugation into 0 to 20 S, 20 to 30 S, and 30 to 50 S fractions. This produces an analogous effect of dividing the 45 S preribosomal RNA into three regions as far as the ribose methylation is concerned; the first region represents 17% of the 45 S RNA from the 5′-end (0 to 20 S), the second region represents 31% of the 45 S RNA from the middle segment (20 to 30 S), and the third region represents 52% of the 45 S RNA from the 3′-end (30 to 50 S). The methylated nucleotide patterns (both base and ribose) of these fractions were analyzed and compared with the patterns of the ribosomal RNAs. The ribose methylation patterns of the 0 to 20 S fraction resembled more closely those of 18 S ribosomal RNA, and the ribose methylation patterns of the 30 to 50 S fraction resembled more closely those of 28 S ribosomal RNA. A marker alkali-stable trinucleotide, probably UmpGmpU, known to be present only in 28 S ribosomal RNA, was found to be labeled and concentrated in the 30 to 50 S fraction under these conditions. Preferential inhibition by 0·5 m -KCl of the methylation of nascent RNA sedimenting as the lower molecular weight fraction led to the production of methylated patterns resembling more closely those of 28 S ribosomal RNA. It is concluded from these data that 18 S ribosomal RNA is located nearer to the 5′-end and 28 S ribosomal RNA is nearer to the 3′-end of the 45 S preribosomal RNA. The results also demonstrate the validity and utility of these procedures for the study of ribosomal RNA synthesis in vitro .

A rat-mouse hybrid cell line was examined for the presence of ribosomal RNA and ribosomal proteins from both parents. As demonstrated by banding of centromeric heterochromatin, the hybrid cell line contained most of the mouse genome and at least 13 rat chromosomes. The ability of rat, but not mouse, ribosomes to dimerize was used to show that the hybrid contained both rat and mouse 28S ribosomal RNA. Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins indicated the presence of both rat and mouse ribosomal proteins.

The metaphase chromosomes of Notophthalmus (Triturus) viridescens have been studied by C-banding and in situ hybridization. The chromosomes show the pericentric C-banding seen in many organisms and in addition have interstitial C-bands located a short distance from the pericentric C-bands on each chromosome arm. A few C-bands are seen in telomeric regions. Regions which hybridize in situ with 18S and 28S ribosomal RNA were found on three chromosome pairs. The animals studied fell into three groups with respect to which of the six possible sites showed detectable hybridization with 18S and 28S RNA. Individual animals differed not only in the pattern of in situ hybridization of ribosomal RNA but also in the number of ribosomal RNA cistrons in the genome as measured by saturation hybridization on purified DNA. In situ hybridization showed five pairs of chromosomes which contained DNA complementary to 5S RNA. The four pairs of subtelocentric chromosomes in the N. viridescens karyotype all have 5S DNA in the pericentric regions. The fifth cluster of 5S DNA is in the middle of one arm of the chromosomes in one of the two smallest submetacentric pairs in the genome. The five sites of 5S DNA differ markedly in the level of in situ hybridization with 5S cRNA.

The minor RNA components in large ribosomal subunits of rat liver were analyzed by gel electrophoresis. Quantitative analysis showed that these minor components, whose apparent molecular weights ranged from 8.48×105 to 11.4×105, represented 10 to 13% of the total high-molecular-weight ribosomal RNA. It was elucidated that they were not artifacts arose during the cell homogenization, subcellular fractionation, RNA preparation and gel electrophoresis. Labeling experiment in vivo showed that they were preferentially present in “old” ribosomes. It was predicted that these minor components were the intermediates in the degradation of 28S ribosomal RNA in vivo. In the regenerating liver, where the degradation of proteins was reported to be blocked, the relative amount of the minor RNA components was decreased to about a half of that of normal liver. There was, however, no increase in their relative amount in the long-term starved rat liver, where RNA degradation should be going very rapidly.

The 5' and 3' terminal T1 oligonucleotides of 32p-labelled B. mori 18S ribosomal RNA were isolated by a two dimensional electrophoretic (diagonal) technique. Nucleotide sequence analysis showed that the 3' terminal fragment, (G)AUCAUUAOH, is identical to that previously obtained from the 18S rRNA of several other eukaryotic species. The sequence of the B. mori 5' terminal fragment is pUCCUCG.

Precise localisation of the structural gene for the S18 ribosomal protein has been completed. The rpxR gene maps at minute 84 on the bacterial chromosome between the cycA and pyrB markers.

Kinetics of incorporation of radioactive guanine into 16 S and 23 S ribosomal RNA were determined in Escherichia coli under two different growth conditions at 37 °C—with glucose, and with succinate. The times required to produce the individual molecules were determined by analysis of the labeling kinetics. Three main inferences are drawn. (1) Ribosomal RNA transcription proceeds at about 84 nucleotides per second with glucose and about 48 nucleotides per second with succinate. (2) The total magnitude of rRNA synthesis in the cell depends strongly on the carbon source, as does the transcription rate. However, the transcription rate does not vary sufficiently to account for the different rates of rRNA synthesis. The overall magnitude must therefore be controlled by the rate of chain initiation. (3) 23 S rRNA is assembled by a straightforward transcription process and not by the linking together of two 16 S-sized molecules.

DNA from spermatids and 12-day-old larvae of Ascaris lumbricoides was isolated and used as a source of germ line and somatic DNA. Ribosomal RNA from Ascaris larvae with molecular weights of 0.76 × 106 for the smaller and 1.42 × 106 for the larger unit was labeled in vitro and hybridized to germ line and somatic DNA. The saturation values revealed that about 0.2% of each DNA is complementary to 18S and 28S rRNA, which is equivalent to approximately 350 and 255 ribosomal genes per haploid spermatid and larval genome, respectively.

HeLa cell “5.8 S” ribosomal RNA was digested with T 1 ribonuclease and the digestion products were characterized. In particular several hexa-, or larger, oligonucleotides were well fractionated by T 1 ribonuclease plus alkaline phosphatase fingerprints. The sequences of these large products were determined. The same large products were identified in fingerprints of “native” 28 S RNA, that is, 28 S RNA to which 5.8 S RNA is attached. The products were demonstrably absent in fingerprints of heat-denatured 28 S RNA, which lacks the 5.8 S fragment. The oligonucleotides were present in fingerprints of 32 S RNA, whether previously heated or not. One of the largest 5.8 S oligonucleotides contains an alkali-stable (2′- O -methylated) dinucleotide, Gm-C. This product was identified in fingerprints of methyl-labelled 45 S RNA. These findings prove that the 5.8 S ribosomal sequence is present within HeLa cell ribosomal precursor RNA. In addition to the methylated nucleotide, two pseudouridylate residues were discovered in HeLa cell 5.8 S RNA.

The 16S ribosomal RNA of the Euglena gracilis chloroplast has been characterized in terms of its two-dimensional electrophoretic "fingerprint" (T1 ribonuclease). Results show it to be a typically prokaryotic 16 S rRNA. By the present criterion, different chloroplasts are shown to be related to one another and at least distantly to blue-green algae and perhaps to Bacillaceae. These results argue in favor of an endosymbiont origin of the chloroplast.

Circular dichroism (CD) and infrared (IR) spectroscopic analyses were performed of 26 and 18S yeast ribosomal RNA's (rRNA) and their specific complex, 30S RNA. The molecular ellipticity coefficients [thota] of 18, 26, and 30S rRNA's were 2.72, 2.63, and 2.65X10(4) degree-cm2/decimole at 264 nm, respectively. The base-pairing contents of 18, 26, and 30S rna's determined by iC), and 70% (32% AU, 38% GC), respectively. These results suggest that 18 and 26S rRNA have very similar secondary structures, and that 30S rna may have a slightly higher base-pairing content than the estimated sum of those of 18 and 26S rRNA's. The biological significance of this phenomenon is discussed in this report.

The insertion mutations I15 and I16 in the ribosomal protein gene cluster at 64 min in Escherichia coli are identified as IS1 and IS2 using electron microscope heteroduplex analysis.

Chloroplast DNAs of higher plants have been found to contain two chloroplast ribosomal RNA genes. The base sequences of these chloroplast ribosomal RNA genes in chloroplast DNAs have been studied by molecular hybridization. The results have shown that these genes have undergone little divergence in the evolution of either monocotyledonous or dicotyledonous plants.

A method to localize individual proteins on the surface of the ribosomal subunit was developed. The method uses specific immunoglobulins G against proteins under examination. The antibodies are combined with the ribosomes to give rise to ribosome dimers linked by the bivalent antibody. The Fab arm of the Y shaped antibody points to the position of the protein the antibody was prepared against. Using this method, two ribosomal proteins (L1, L19) ‡ were located on two defined shapes of the 50 S ribosomal subunit.

Further studies were performed on the sequence of addition of proteins to 16 S RNA during the in vitro reconstitution of 30 S ribosomal subunits from Escherichia coli. Direct binding of protein S17 to 16 S RNA was studied in detail, and the following results were obtained: (a) under reconstitution conditions, a maximum of approximately 1 mole of S17 is bound per mole of 16 S RNA, either alone, or in the presence of all other 30 S proteins; (b) S17 binds only to 16 S RNA and not to 23 S RNA; and (c) radioactive S17–16 S RNA complexes are directly converted (without dissociation) to 30 S subunits by the addition of excess unlabeled total 30 S proteins. From these results, we conclude that the binding of S17 to 16 S RNA is specific. We have also determined the positions of S15, S16, S17, and S12 in the assembly map and have clarified subsequent interactions depending on these proteins. A revised assembly map is presented which incorporates the additional information obtained from these experimental results.