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The yeast virus, ScV, is communicated only by mating. It has two separately encapsidated dsRNAs. One of these, L, codes for the major capsid polypeptide. The other, M, codes for a polypeptide toxic to yeasts without ScV-M particles. Defective interfering particles containing fragments of M (S) displace ScV-M when they arise. We have shown that five independently isolated S dsRNAs are all derived by internal deletion of M. The 3' ends of all the ScV dsRNAs are markedly heterogeneous. For instance, half of the first 35 nucleotides at one 3' end of M and S are variable. Conserved sequences at the 3' ends of M and S are AAACACCCAUCAOH and AUUUCUUUAUUUUUCAOH. Conserved sequences at the 3' ends of L are UAAAAAUUUUUCAOH and AAAAAUXCAOH, where X is variable. We propose that the sequence AUUUUUCAOH is a recognition sequence for the capsid-associated single-stranded RNA polymerase activity. Since all the viral RNAs have pppGp 5' termini, their 3' termini probably extended one nucleotide beyond the terminal pppGp.

Recombinants with a centrally located crossover point were selected from crosses between poliovirus type 1 strains and intertypic (type 3/type 1) recombinants. Two such recombinants were characterized in some detail. In one of them (v1/a1-6), the 5' half of the genome was derived from a virulent type 1 strain, while the 3' half came from an attenuated type 1 strain. The genome of the other recombinant (a1/v1-7) had the reverse organization, with the 5' and 3' halves being derived from the type 1 attenuated and virulent strains, respectively. As deduced from the RNase T1 oligonucleotide maps, the a1/v1-7 genome also had a relatively short centrally located insert of the poliovirus type 3 origin. Both recombinants exhibited ts phenotypes. The RNA phenotypes of the recombinants corresponded to that of the parent donating the 3' half of the genome, v1/a1-6 and a1/v1-7 expressing RNA- and RNA +/- characters, respectively. Despite being a ts RNA- virus, v1/a1-6 proved to be neurovirulent when injected intracerebrally into Cercopithecus aethiops monkeys, although it exhibited a somewhat diminished level of pathogenicity as compared to its virulent type 1 parent. Recombinant a1/v1-7 behaved as an attenuated strain. These data supported our previous conclusion drawn from the experiments with intertypic poliovirus recombinants that the attenuated phenotype of poliovirus depends largely on the structure of the 5' half of its genome, although mutations of the 3' half may alleviate the virulence of the virus to a degree.

A 4 month old child was immunized with a vaccine containing the Sabin live attenuated vaccine strains of all three serotypes of poliovirus. The antigenic and molecular evolution of the Sabin strain of poliovirus type 3 was then followed throughout the entire period of virus excretion. Novel strains appeared at 8, 42 and 52 days post-vaccination and were the products of both intertypic recombination between type 2 and type 3 poliovirus in regions of the genome coding for non-structural proteins and of point mutations in the region coding for the structural proteins. Excretion of virus continued for 73 days. All strains examined reacted with all monoclonal antibodies specific for the main immunodominant antigenic site of type 3 poliovirus, but variation was observed at other, immunorecessive sites. These findings have possible implications for the evolution of the virus in vaccinees or in epidemics and are consistent with the known antigenic stability of the virus.

The nucleotide sequence of a low molecular weight RNA coded by bacteriophage T4 (and previously identified as species alpha) has been determined. The molecule is of particular biological interest for its associated biosynthetic properties. This RNA is 76 nucleotides in length, contains eight modified bases, and can be arranged in a cloverleaf configuration common to tRNAs. The anticodon sequence is UGU, which corresponds to the threonine-specific codons ACA G. The nucleotide sequence was determined primarily by nearest-neighbor analysis of RNA synthesized in vitro using [alpha-32P]nucleoside triphosphates. Using the single-strand specific nuclease S1, two in vivo labeled half-molecules were generated and analysed. This information together with restrictions imposed by nearest-neighbor data, provided a unique linear sequence of nucleotides with the features of secondary structure common to tRNA molecules.

Three different conformers of rat liver 5S ribosomal RNA were investigated by partial nuclease cleavage technique using S1 nuclease and cobra venom endoribonuclease (CVE) as conformational probes. Urea-treated and renatured 5S RNA co-migrate on non-denaturing gels, but exhibit distinct differences in their nuclease cleavage patterns. The most prominent differences in S1 nuclease and CVE accessibility of these conformers are located in region 30-50 and around nucleotides 70 and 90. The third form of 5S RNA with higher electrophoretic mobility was generated by EDTA treatment. The cleavage patterns of this 5S RNA conformer are similar to that characteristic for the renatured 5S RNA. The results demonstrate the difference in secondary structure and possibly different tertiary base-pairing interactions of 5S RNA conformers.

A guanine insertion enzyme (tRNA transglycosylase) was purified to a homogeneous state from Escherichia coli B by ammonium sulfate fractionation and DEAE-cellulose, DEAE-Sephadex A-50, phosphocellulose, and Sephadex G-200 column chromatographies. The molecular weight of the enzyme, which appeared to be a single polypeptide, was 4.6 X 10(4) by sodium dodecyl sulfate gel electrophoresis. The enzyme catalyzes exchange of guanine with guanine located in the first position of the anticodon of tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp, but unlike the enzymes isolated from rabbit reticulocytes and Ehrlich ascites tumor cells it does not catalyze the exchange of guanine with queuine (7-(3,4-trans-4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanine) present in these tRNAs. The pH optimum of the reaction was 7.0, and the pH1 value was 4.6 to 4.8. The reaction required Mg2+ ion. 7-Methylguanine inhibited guanine insertion, but the other purine analogues tested were not inhibitory and could not replace guanine.20

One of the two major species of brewer's yeast tRNA threonine (tRNA Thr 1) has been purified by countercurrent distribution followed by two chromatographic steps (respectively on a Sepharose 4B and a BD-cellulose column). Complete digestion with pancreatic and T1 RNases and a partial hydrolysis with T1 RNase followed by the isolation and determination of the nucleotide sequences of the resulting fragments permitted the derivation of its primary structure. tRNA Thr 1 is in fact a mixture of two subspecies differing only by a A49-U65 base pair in 50 per cent of the molecules which is replaced by a G49-C65 pair in the other 50 per cent. These two subspecies consist of 76 nucleotide residues including 14 minor nucleotides. They show a characteristic m3C at the 3'terminal end of the anticodon loop, an anticodon I-G-U followed by t6A and C48, uncompletely modified (50 per cent) to m5C within the 5 nucleotides long extra-arm. The minor nucleotides m2G m2 2G are located at positions in which they generally occur in the tRNA structures as does m1A within the T-psi-C loop.

A double-labeling procedure for sequence analysis of nonradioactive polyribonucleotides is detailed, which is based on controlled endonucleolytic degradation of 3'-terminally (3H)-labeled oligonucleotide-(3') dialcohols and 5"-terminal analysis of the partial (3H)-labeled fragments following their separation according to chain length by polyethyleneimine- (PEI-)cellulose TLC and detection by fluorography. Undesired nonradioactive partial digestion products are eliminated by periodate oxidation. The 5'-termini are assayed by enzymic incorporation of (32p)-label into the isolated fragments, enzymic release of (32p)-labeled nucleoside-(5') monophosphates, two-dimensional PEI-cellulose chromatography, and autoradiography. Using this procedure, as little as 0.1 - 0.3 A260 unit of tRNA is needed to sequence all fragments in complete ribonuclease T1 and A digests, whereas radioactive derivative methods previously described by us1-4 required 4 - 6 A260 units.

We have isolated a bacterial amber mutation (nadam) that is suppressed by the tyrosine inserting suppressor su+3 but not by the glutamine (su+2, su+3 A1, su+3 G82 and su+3 A1G82), serine (su+1) and leucine (su+6) inserting suppressors. The su+7 suppressor which inserts glutamine and tryptophan also suppresses this mutation indicating that tryptophan, in addition to tyrosine, is accepted at the site of amber mutation. We have used this amber mutation to search for revertants of the su+3 glutamine mischarging mutants su+3 A1, su+3 G82 and su+3 A1G82 that are able to insert tyrosine at the site of amber mutation. Two types of revertants were found in the case of su+3 A1. One type corresponding to the true revertant A1 leads to G, and the other to the second site revertants C81 leads to U (A1U81). The A1U81 revertant has been shown to insert both glutamine and tyrosine at the site of amber mutation. Only true revertants (G82 leads to A) were obtained when su+3 G82 was analyzed. No revertants were obtained in the case of the su+3 A1G82. These results are discussed in relation to aminoacyl-tRNA recognition.

We have employed S1 nuclease to probe the structure of an intermediate in tRNA biosynthesis available only in radiochemical purity. The dimeric precursor to tRNAGln and tRNALeu from bacteriophage T4 was digested with the single-strand specific nuclease, and the products of the reaction were compared with the S1 digestion products of the mature cognate tRNA'S. Quantitation and sequence analysis of the products revealed that the location and accessibility of S1 cleavage sites in the precursor were substantially identical with those in the mature forms. Based on these conclusions, it is argued that the dimer is comprised of two domains in which the specific features of both secondary and tertiary conformation closely resemble those found in the mature molecules; at the same time we noted small but apparently significant differences in certain regions of the molecule which may reflect signals for various maturation events. Finally, we have determined that the sites of precursor cleavage by RNase P, the endonuclease which generates the mature 5' termini of these tRNAs, were completely inaccessible to S1 digestion.

Borate gel chromatography was used to separate internal oligonucleotides containing N6-methyladenosine (m6A) from methylated 5'-terminal oligonucleotides of HeLa cell polyadenylylated mRNA. N6-Methyladenosine occurs primarily in two sequences, -G-m6A-C (70%) and -A-m6A-C-(30%). The nucleoside immediately following cytidine may be uridine, cytidine, or adenosine, while guanosine as well as other nucleosides occupy subsequent positions. Each of the four positions preceding the -(G or A)-m6A-C- sequence may be occupied by a pyrimidine or a purine ribonucleoside. Since on a random basis all possible sequences containing -(G or A)-A-C-(U or C or A)- could occur once per 43 nucleotides whereas there is only one m6A residue per thousand nucleotides, then either (1) not all potential sites are methylated, (2) there are multiple unique sequences perhaps methylated by several different enzymes, or (3) there are other unrecognized discriminating factors. The possibility that methylation of adenosine occurs exclusively in the region close to the 5' terminus of the mRNA was considered. However, such a localization was excluded since the majority of m6A residues were not found in 4 to 6S 5'-terminal fragments isolated by borate gel chromatography.

RNAase P has been partially purified from three thermosensitive strains of E. coli and the thermal inactivation characteristics of each preparation have been determined. The RNAase P preparations from two of these mutant strains, ts241 and ts709, and the wild-type strain have been separated into RNA and protein components. Various mixtures of the reconstituted components have been checked in vitro for complementation of their thermal sensitivity properties. The protein component of RNAase P from ts241 and the RNA component of RNAase P from ts709, respectively, account for the thermal sensitivity of the rnaase P from the two strains. The amount of the RNA component of RNAase P is lower in ts709 than in ts241 or the wild-type parent, 4273. RNAase P partially purified from a revertant of the third mutant strain, A49, which maps at or near the ts241 mutation, has an altered charge when compared to the RNAase P from the parent strain, BF265. We conclude that mutations which affect either the protein or RNA component of RNAase P can confer thermal sensitivity on the enzyme both in vivo and in vitro.

We present a simple program running on a pocket computer allowing us to determine the order of importance of the wavelengths (regarding the accuracy of the results) in spectral analysis and to evaluate the absolute errors made on the determination of the concentrations of the constituents. We have applied this programme to the determination of the composition of a tetraribonucleotide mixture and have compared our results with other results published.

The following contribution summarizes our most recent results concerning analysis of the influenza A, B and C virus genomes. In addition, we present data on the extent of genetic variation of H1N1 influenza viruses isolated during the months following the 1977 outbreak of H1N1 influenza in China and Russia. A detailed description of these results is published elsewhere.

The guanine insertion enzyme isolated from Escherichia coli (tRNA transglycosylase) catalyzed the incorporation of bases of Q (queuosine) precursors into E. coli undermodified tRNAAsn and tRNATyr. These bases of Q precursors were inserted in the first position of the anticodon of tRNASn and tRNATyr, replacing guanine originally located in that position. This is a novel type of post-transcriptional modification, inserting a modified base into the polynucleotide chain by cleavage of the N--C glycoside bond without breakage of the phosphodiester bond. One of the bases of Q precursors, 7-(aminomethyl)-7-deazaguanine, was found in the acid-soluble fraction of E. coli cells, supporting the conclusion that formation of Q, 7-(3,4-trans-4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanosine, in tRNA in vivo actually proceeds by the tRNA transglycosylase reaction.

The reaction of 14C methyl-labelled HeLa cell 28 S ribosomal RNA with sodium bisulphite was studied. Using conditions under which 30% of the total cytidine residues were de-aminated to uridine, the reactivities of individual cytidine residues near particular methylation sites differed widely; some underwent almost quantitative reaction, some showed intermediate reactivity and others were almost inert. The possible value of this method as a conformational probe for ribosomal RNA is discussed.

Naturally occurring differences in the nucleotide sequences of 5.8S ribosomal ribonucleic acids (rRNAs) from a variety of organisms have been used to study the role of specific nucleotides in the secondary structure and intermolecular interactions of this RNA. Significant differences in the electrophoretic mobilities of free 5.8S RNAs and the thermal stabilities of 5.8S--28S rRNA complexes were observed even in such closely related sequences as those of man, rat, turtle, and chicken. A single base transition from a guanylic acid residue in position 2 in mammalian 5.8S rRNA to an adenylic acid residue in turtle and chicken 5.8S rRNA results both in a more open molecular conformation and in a 5.8S--28S rRNA junction which is 3.5 degrees C more stable to thermal denaturation. Other changes such as the deletion of single nucleotides from either the 5' or the 3' terminals have no detectable effect on these features. The results support secondary structure models for free 5.8S rRNA in which the termini interact to various degrees and 5.8S--28S rRNA junctions in which both termini of the 5.8S molecule interact with the cognate high molecular weight RNA component.

We have used a combination of techniques of DNA and RNA sequence analysis to determine the nucleotide sequence of the portion of simian virus 40 DNA preceding and encoding the 5' end of mRNA for the structural protein VP2 of simian virus 40. Comparison of the sequence with those found in polyadenylated RNA in the cytoplasm of infected cells RNA shows that the transcript of sequences preceding the structural gene is more abundant than the transcript containing the codons for the protein. Between the abundant transcript of sequences preceding the coding region and the less abundant transcript of the coding region there is a short sequence whose transcript is not detected.

The methionine acceptor activity of a crude tRNA from bakers' yeast was resolved into two peaks (I and II) by column chromatography on DEAE-Sephadex A-25 with a 1 M phosphate system. Methionine tRNA from peak II was not formylated by E. coli methionyl-tRNA transformylase [EC 2.1.2.9.] after being charged with methionine, whereas that from peak I was formylatable under the same conditions. A substantial amount of unlabelled methionine tRNA, tRNAMetm, was highly purified from the peak II fraction by successive chromatographic procedures. The purified tRNAMetm was digested with pancreatic ribonuclease A [EC 3.1.4.22] and ribonuclease T1 [EC 3.1.4.8]. The digestion products were isolated into individual components and completely sequenced. The results of sequence analysis of the two RNase digests were in good agreement and indicated that the chain length of this tRNA is 76, including 13 modified nucleotides. These oligonucleotide fragments can be constructed into a unique total sequence, assuming a few conventional features of clover leaf structure for the tRNA was established by analyses of partial digestion products with RNase T1, as reported in the accompanying paper.

Two protein, VPgA and VPgB, are covalently bound to the virion RNA of encephalomyocarditis (EMC) virus. Their molecular weightrs, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate are 10,000 and 8,000, respectively. A study of nucleotide-peptides isolated from VPg-RNA compound has shown that VPgA is bound to the 5'-terminal nucleotide of RNA by a phosphodiester bond. The 5'-terminal nucleotide of RNA is uridylic acid. It is the hydroxy group of the Tyr residue of VPgA that is involved in the formation of the linkage with RNA. VPgB-RNA seems to be similar to VPgA-RNA both in the structure of the RNA-protein linkage and localization of VPgB on RNA.

A precursor of 5S ribosomal RNA from Bacillus subtilis (p5A rRNA, 179 nucleotides in length) is cleaved by RNase M5, a specific maturation endonuclease which releases the mature 5S rRNA (m5, 116 nucleotides) and precursor fragments derived from the 5' (21 nucleotides) and 3' (42 nucleotides) termini of p5A rRNA. Previous results (Meyhack, B., et al. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3045) led to the conclusion that recognition elements in potential RNase M5 substrates mainly reside in the mature moiety of the precursor. Limited digestion of p5A rRNA with RNase T1 permitted the isolation of a number of test substrates which contained both precursor-specific segments and were unaltered in the immediate vicinity of the cleavage sites, but which differed in that more or less extensive regions of the mature moiety of the p5A rRNA were deleted. Tests of the capacity of these partial molecules to serve as substrates for RNase M5 indicate clearly that the enzyme recognizes the overall conformation of potential substrates, neglecting only the double-helical "prokaryotic loop" (Fox, G.E., & Woese, C.R. (1975) Nature (London) 256, 505).

Purified 15 S globin mRNA-protein (mRNP) complexes obtained by EDTA dissociation of duck reticulocytes polyribosomes were digested with the calcium dependant Staphylococcus aureus nuclease (EC 3. 1. 4. 7.). 25% of the globin mRNA sequences were resistant to extensive nuclease digestion as determined by TCA precipitation of the digested 15 S particles labelled in vivo with tritiated uridine. Polyacrylamide gel electrophoresis of the RNA from nuclease digested 15 S particles showed that the protected oligoribonucleotides were distributed into two distinct size classes of 25,000 and 12,000 MW. Comparison between in vitro iodine-labelled 9 S globin mRNA extracted from Staphylococcal nuclease digested 15 S mRNP particles was carried out by fingerprinting. Mapping of T1 ribonuclease digests by high-voltage electrophoresis and homochromatography showed that specific oligoribonucleotides were protected against nuclease attack by proteins of the 15 S mRNP.

In the DNA of higher vertebrates the dinucleotide CG is unique in two respects: it occurs far less frequently than would be expected on the basis of the content of cytosine and guanine in a given DNA segment ("CG suppression") and it contains predominantly 5-methyl-cytosine, the only modified nucleotide common in vertebrate DNA. Here we point out the existence of CG clusters, i.e. localized lapses in the usual CG suppression, in two categories of DNA segments from vertebrates: around the polymorphic exons of major histocompatibility complex (MHC) genes and in the 5' regions of certain other genes. These observations contradict the recent suggestion that CG frequency is uniform over long contiguous segments of DNA containing several genes. A model for the origin of these CG clusters as a consequence of regional demethylation of germline DNA is supported by analysis of other sequence features of these regions as well as by previously published data on the methylation status in sperm DNA of two of these CG-rich regions.

Intratypic recombinant polioviruses were isolated from cells that were coinfected with two temperature-sensitive (ts) mutants of poliovirus type 1, ts035Gr and ts247. After phenotypic characterization of these recombinants, their proteins were studied by polyacrylamide gel electrophoresis, and their genomes were analyzed by RNase T1 fingerprinting and partial nucleotide sequencing. Segregation of specific phenotypic and biochemical characteristics inherited from the parental viruses demonstrated that crossing-over could occur in at least four distinct regions of the genome. Possible mechanisms for recombination are discussed.

5 S RNA was isolated from Saccharomyces cerevisiae grown in the presence of 32P-phosphate and digested with nuclease S1, a single-strand specific nuclease. Two different procedures were employed to determine the sites of attack on the RNA. First, 5 S RNA was isolated from nuclease S1 digests, digested to completion with ribonuclease T1, and then 'fingerprinted' by two-dimensional electrophoresis. Quantitation of each of the characteristic RNAase T1-derived oligonucleotides was employed to determine the relative susceptibility of various regions of the molecule to nuclease S1. A second procedure to define nuclease S1-susceptible sites in the molecule employed polyacrylamide gel electrophoretic fractionation of nuclease S1 digests followed by identification of the nucleotide sequences of the released RNA fragments. Both procedures showed that the region of the molecule between residues 9 and 60 was most susceptible to nuclease S1, with preferential cleavage occurring between residues 12-25 and 50-60. These results are discussed in relation to a proposed model for the secondary structure of yeast 5 S RNA.

The 32P-labelled genomes of poliovirus type 1, 2 and 3 have been digested with RNase T1 and the products separated by two-dimensional gel electrophoresis. All three fingerprints differ in the separation pattern of the large oligonucleotides. The molar yields of the large RNase T1-resistant oligonucleotides of type 1 and type 2 RNA of poliovirus RNA are close to one. By comparing the yields of these oligonucleotides to the amount of RNA from which they originated, the chain length of type 1 poliovirus RNA was found to be 7851 +/- 567 nucleotides (mol. wt. 2.66 +/- 0.19 x 10(6) and that of poliovirus type 2, 8181 +/- 578 nucleotides (mol. wt. 2.77 +/- 0.19 x 10(6). The chain length of two defective interfering particle (DI) RNAs of poliovirus type 1 were determined to be 7042 +/- 999 nucleotides for DI(1) and 6639 +/- 674 nucleotides for DI(2).

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.

The complete nucleotide sequences of the various precursor 5S RNA species occurring in Bacillus licheniformis have been elucidated. The B. licheniformis precursors contain a 5'-precursor-specific segment of 95 nucleotides which is four times as long as the corresponding segment of the p5S RNAs from the closely related strains B. subtilis (Sogin, M.L., Pace, N.R., Rosenberg, M., Weissman, S.M. (1976) J. Biol. Chem. 251, 3480-3488) and Bacillus Q (Stiekema, W.J., Raué, H.A., Planta, R.J. (1980) Nucl. Ac. Res. 8, 2193-2211). However, fourteen of the sixteen nucleotides at the 5'-end are identical in the precursors from all three strains. These conserved nucleotides can form a stem and loop structure which is likely to play an important role in the biosynthesis of 5S RNA. Extension secondary and tertiary structure is present in the 5'-precursor-specific segment as concluded from the results of digestion with RNAase T1 both of the isolated segment and the intact precursors. No sequence homology exists between the 3'-precursor-specific segments of the B. licheniformis precursors and those of the other two strains except for a stretch of U residues at the 3-terminus. This stretch of U residues is not immediately preceded by a hairpin loop, however, as expected for a transcription termination signal (20). The question whether the precursors have already undergone processing at the 3'-end, therefore, remains open. The total number of genetically distinct precursor species in B. licheniformis is at least five and at most ten. Most likely each ribosomal RNA cistron produces a separate p5S RNA as is also the case in Bacillus Q.

Mitochondrial tRNAPhe from Saccharomyces cerevisiae isolated by two-dimensional gel electrophoresis was sequenced by fingerprinting uniformly labeled 32 P-tRNA as well as by 5'-end postlabeling techniques. Its sequence was found to be: pG-C-U-U-U-U-A-U-A-G-C-U-U-A-G-D-G-G-D-A-A-A-G-C-m22G-A-U-A-A-A-phi-U-G-A-A-m1G-A-phi-U-U-A-U-U-U-A-C-A-U-G-U-A-G-U-phi-C-G-A-U-U-C-U-C-A-U-U-A-A-G-G-G-C-A-C-C-A. The secondary structure we propose, in order to maximize base pairing in the phiC stem and to allow tertiary interaction between G15 and C46, excludes U50 from base pairing giving a bulge in the phiC stem. No conclusion can be drawn concerning the endosymbiotic theory of mitochondria evolution by comparing the primary structure of mt. tRNAPhe with other sequenced tRNAsPhe. This mt.tRNAPhe lacks some of the structural elements reported to be involved in the yeast cytoplasmic phenylalanyl-tRNA ligase recognition site and cannot be aminoacylated by purified yeast cytoplasmic phenylalanyl-tRNA ligase.

The use of a highly specific radioimmunoassay and of HPLC permitted us to confirm the occurrence of 2'-5' oligoadenylates [p chi (A2'p5')nA] in several strains of Escherichia coli. Cellular concentrations of 2'-5' oligoadenylates ranged from 50 nM to 300 nM. The mixture of 2'-5' oligoadenylates consisted primarily of pppA2'p5'A, pA2'p5'A,A2'p5'Ap and A2'p5'A under normal conditions of growth. None of them activated RNase L. Infection of the bacteria with the single-stranded DNA phage M13 or induction of a heat-inducible, non-lytic mutant of phage lambda led to a significant increase in the total pool of 2'-5' oligoadenylates, paralleling the progressive inhibition of growth. Likewise, the inhibition of protein synthesis with chloramphenicol stimulated the accumulation of 2'-5' oligoadenylates. Furthermore, the inhibition of bacterial growth by either phage or by chloramphenicol brought about a change in the composition of the 2'-5' oligoadenylate pool; 5'-phosphorylated 2'-5' oligoadenylates accumulated and became the major components. The findings indicate a parallelism between the effects of viral infection on the synthesis of 2'-5' oligoadenylates in eukaryotes and similar effects subsequent to phage growth in the bacterium E. coli.

The genetic heterogeneity of five influenza A (H3N2) strains isolated between 1968 and 1977 has been estimated by T1-oligonucleotide fingerprinting of 32P-labeled viral RNA. Assuming that the large T1-resistant oligonucleotides represent a random sample of the viral RNA, the genetic differences observed would affect 0.3 to 10.7% of the RNA positions of the genes studied, depending on the pair of viruses considered. A smaller degree of genetic heterogeneity was observed when six coetaneous viral samples were compared. The distribution of spontaneous mutations among the viral genes was studied by fingerprinting individual RNA segments isolated either by gel electrophoresis or hybridization with plasmids containing influenza-specific DNA sequences. No statistically significant differences were detected in the distribution of mutations among the viral genes studied. The mutation frequency at the hemagglutinin RNA region coding for the HA1 subunit was found to be two times higher than that at the region encoding that HA2 subunit. Our results suggest that the antigenic variability of influenza viruses may be a consequence of a general genetic variability which effects many of the viral genes.

In June of 1977, a new influenza A pandemic was started by strains of the H1N1 serotype. Oligonucleotide fingerprint analysis of the RNA from viruses isolated during the early stage of this pandemic demonstrated that genetic variation among these 1977 strains could be attributed to sequential mutation [Young, J.F., Desselberger, U. & Palese, P. (1979) Cell, 18, 73-83]. Examination of more recent strains revealed that the H1N1 variants that were isolated in the winter of 1978-1979 differed considerably from the H1N1 viruses isolated the previous year. Oligonucleotide and peptide map analysis of the new prototype strain (A/Cal/10/78) suggested that it arose by recombination. It appears that only the HA, NA, M, and NS genes of this virus are derived from the earlier H1N1 viruses and that the P1, P2, P3, and NP genes most likely originate from an H3N2 parent. These data suggest that genetic variation in influenza virus strains of the same serotype is not restricted to mutation alone, but can also involve recombination (reassortment).

The low molecular weight RNA species which have been purified and characterized in this laboratory (T.S. Ro-Choi and H. Busch (1974) in The Cell Nucleus, Vol. 3, pp 151-208; Academic Press, New York) are now of interest because of their suggested role in processing of heterogeneous nuclear RNAs (Lerner, M.R., Boyle, J.A., Mount, S.M., Wolin, S.L., and Steitz, J.A. (1980) Nature, 283, 220-224). A previously uncharacterized RNA, U6 (4.7 S) nuclear RNA, which is 106 nucleotides long, was extracted from Novikoff hepatoma ascites cell nuclei and purified by polyacrylamide gel electrophoresis. The primary nucleitde sequence of U6 RNA was determined by subjecting the RNA to several types of enzymatic digestions and gel-sequencing techniques. The sequence is: (formula: see text). U6 RNA contains three pseudouridylic acid residues, four alkali-stable dinucleotides, two alkali-stable trinucleotides, one m6adenosine, and one m2guanosine and is notable for the high concentration of modified nucleotides in the center of the molecule. U6 RNA has an unusual 5' terminus which has not yet been fully characterized.

Polyacrylamide gel electrophoretic analysis of RNA segments of the arenaviruses Pichinde (Pic) and Tacaribe (Tac) showed them to be distinguishable in that Pic S RNA had a slower electrophoretic mobility than Tac S RNA. The L and S RNA segments of Tac virions were found to have distinct RNase T1 oligonucleotide fingerprints, indicating that they are unique RNA species. The oligonucleotide patterns of the Tac L and S RNAs were also distinct from those of the corresponding Pic RNA segments.

Chemical reactivity of cytosines in 32P-labeled E. coli tRNA1Leu, E. coli tRNAPhe and yeast tRNAPhe before and after aminoacylation was examined by use of a cytosine-specific reagent, semicarbazide-bisulfite mixture. In all the three tRNA species examined, the cytosine residues that were susceptible to the modification were the same in the aminoacylated tRNA and the unacylated tRNA. Only a limited number of the cytosine residues were modifiable: those that occur in the anticodon, the 3'-CCA terminus, the D-loop, and the extra loop. The sites accessible by the reagent are in good agreement with the general three-dimensional structure of tRNA proposed in literature. These results indicate that the gross conformation of these tRNAs does not change on aminoacylation, and consequently favor the view that the T psi C(G) sequence could become exposed in later steps of protein synthesis in order to achieve the binding of aminoacyl tRNA to ribosomes.

A method is described for the isolation of a yeast chromatin fraction highly enriched in ribosomal DNA sequences. In the presence of exogenous yeast RNA polymerase III, this purified chromatin actively synthesizes a set of 5 S ribosomal RNAs all of which have 5'-sequences identical with mature 5 S RNA but which end with a variable number (up to 10) of additional residues at the 3'-terminus. These extra nucleotides are precisely removed by a processing nuclease found in the chromatin supernatant fraction.

Upon digestion of the complex formed from the 23-S ribosomal RNA and the 50-S ribosomal protein L24 of Escherichia coli, two fragments resistant to ribonuclease were recovered; these fragments contained RNA sections belonging to the 480 nucleotides at the 5' end of 23-S RNA. By determining the sequence of 70% of this latter region we were able to localise the sections which, in the presence of the protein, are resistant to ribonuclease. Our results suggest that the region encompassing the 480 nucleotides starting at the 9th nucleotide from the 5' end of 23-S RNA has a compact tertiary structure, which is stabilised by protein L24.

The 5'-terminus of poliovirus polyribosomal RNA is pUp. A candidate for the 5'-terminus of poliovirion RNA was recovered as a compound migrating toward the cathode when 32P-labeled virion RNA was completely digested with ribonucleases T1, T2 and A and analyzed by paper ionophoresis at pH 3.5. Treatment with proteinase K reversed its direction of migration, indicating the presence of protein. Treatment with venom phosphodiesterase liberated all of the radioactivity as pUp, suggesting that poliovirion RNA has a protein-pUp 5'-terminus. Treatment of virion RNA with T1 ribonuclease alone generated a proteinase K-sensitive oligoribonucleotide. Analysis of the oligoribonucleotide using ribonucleases A and U2 showed its structure to be protein-pU-U-A-A-A-A-C-A-G. Digests of replicative intermediate RNA contained sufficient protein-pUp to suggest that this structure is at the 5'-end of most nascent poliovirus RNA molecules. We suggest that a protein-nucleotide structure acts as a primer for initiating synthesis of poliovirus RNA.

Bacterial RNA, Pulse labeled with 32Pi, was digested with pancreatic RNAase. Oligonucleotides containing a triphosphate group at the 5'-hydroxyl, and therefore derived from the original beginning ends of the RNA transcripts, were purified by hydroxyapatite chromatography and analyzed by two-dimensional paper electrophoresis. A broad diversity of species was found, although the distribution among these species was not completely uniform. Possible methods of utilizing these methods in combination with in vitro synthetic techniques are discussed.

Limited digestion of the Escherichia coli 50-S subunit permits the isolation of two large fragments of the 23-S RNA. One arises from the 5' end of the 23-S RNA and contains about 1200 nucleotides. The other arises from the 3' end of the 23-S RNA and contains about 2000 nucleotides. Each of the ten 50-S proteins known to bind specifically and independently to the 23-S RNA were tested for their ability to interact with these two RNA fragments. It was determined that the 5' terminal segment contains the specific binding sites for proteins L4, L20 and L24, whereas the 3' terminal segment contains the specific binding sites for proteins L1, L2, L3, L6, L13, L13 and L23.