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The association between certain cellular RNAs and purified RNA tumor viruses prompted us to examine the possibility that specific host messenger RNAs might also be incorporated into RNA tumor viruses. Using a mouse cell line infected with Friend leukemia virus, T-3-Cl-2, which can be induced to accumulate mouse-globin messenger RNA, we show that mouse-globin messenger RNA sequences are present in viral particles purified from the culture medium of globin-producing cells. These globin messenger RNA sequences are absent from viral particles derived from T-3-Cl-2 cells that are not producing globin messenger RNA. Virus-associated globin messenger RNA sequences sediment in association with the 60S viral RNA complex as well as in free, 9S form. However, under mild denaturing conditions which result in the conversion of viral 60S RNA to 30S and smaller forms, all the globin sequences sediment as 9S RNA. Appropriate control experiments indicate that the virus-associated globin messenger RNA is resistant to degradation by exogenous ribonuclease; that exogenously added globin messenger RNA does not become associated with the 60S viral RNA complex; and that globin messenger RNA can be detected in virions derived from cells both induced for and constitutively synthesizing globin messenger RNA.

We have used purified mouse immunoglobulin light chain mRNA and synthetic DNA which is complementary to it to assess the reiteration frequency of gene sequences corresponding to the κ constant region of the mouse immunoglobulin light chain. These studies indicate that the constant region sequence is represented only two to three times per haploid mouse genome, a finding that rules out a simple stringent germ line mechanism which would require the constant region sequence to be represented hundreds if not thousands of times. Hybridization studies involving 125 I-labeled myeloma light chain mRNA yield interesting results which may eventually permit us to distinguish between the remaining somatic mutation and recombinational germ line hypotheses. These results reveal a major component of relatively unique frequency and a minor component with a reiteration frequency of approximately 30 to 50 copies per haploid genome. As discussed, these results do not permit us to distinguish unambiguously between a germ line model and a type of somatic mutation model that permits germ line genes corresponding to each κ subgroup. The results do, however, clearly rule out the existence of thousands of variable region sequences so closely related to the MOPC-41 V-region as to permit extensive stable cross-hybridization.

This collaborative publication is a product of the Cold Spring Harbor Symposium of 1963. The authors discuss the state of the field in areas of known factors in messenger efficiency of synthetic polynucleotides, coding ratios, the current "code word dictionary," and synthesis of nucleotides. Tables and figures are included.

RNA from the encephalomyocarditis virus directs the cell-free synthesis of several discrete, high molecular weight proteins. The largest of these have molecular weights of approximately 110,000, 82,000, 73,000, 61,000 and 44,000 Daltons. In addition, tryptic digestion of the in vitro products gives rise to a number of peptides corresponding to those derived from the viral capsid. The data suggest that approximately one-third of the information encoded by the EMC genome is translated in vitro as a single polypeptide chain, that this translation proceeds in an appropriate phase, and that portions of the genome corresponding to structural proteins of the virus are translated.

A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described. The method depends upon annealing poly(adenylic acid)-rich mRNA to oligothymidylic acid-cellulose columns and its elution with buffers of low ionic strength. Biologically active rabbit globin mRNA has been purified by this procedure and assayed for its ability to direct the synthesis of rabbit globin in a cell-free extract of ascites tumor. Inasmuch as various mammalian mRNAs appear to be rich in poly(adenylic acid) and can likely be translated in the ascites cell-free extract, this approach should prove generally useful as an initial step in the isolation of specific mRNAs.

A cell-free system derived from Krebs II ascites tumor has been used to assay biologically active mRNA for myeloma (MOPC-41) light chain during its purification by oligothymidylate-cellulose chromatography and sucrose gradient centrifugation. The purified mRNA directs the synthesis of a product that yields tryptic peptides corresponding to those derived from authentic myeloma protein and that forms a specific immunoprecipitate with antibody directed against the MOPC-41 protein. The fact that the light-chain mRNA anneals to oligothymidylic acid-cellulose suggests that it, like several other eukaryotic mRNAs, contains a region rich in adenylic acid residues. The most active fractions of light-chain mRNA, representing about 0.1% of the RNA originally extracted from membrane-bound myeloma polysomes, sediment as a discrete peak with an s 20, w of about 13, roughly corresponding to an RNA molecule containing 850 bases. The results suggest that the light-chain mRNA is monocistronic and that it contains about 200 more bases than would be necessary to encode the variable and constant regions of a single light-chain molecule.

The synthesis of two classes of late T7 proteins appears to be directed by several polycistronic messenger RNAs. To understand how they are translated in appropriate order during phage infection, we have isolated late mRNA from T7-infected cells and mRNA that was synthesized in vitro with the T7 RNA polymerase and T7 DNA. Using a purified cell-free system in which ribosomal interaction with the message is restricted to the authentic phage initiation sites, we find that late T7 mRNA directs the synthesis of two initiation dipeptides, formylmethionine-alanine and formylmethionine-serine. These dipeptides account for 80% of the incorporated fMet and are synthesized in systems derived from both uninfected and infected female Escherichia coli cells. However, ribosomes isolated from T7-infected female cells apparently contain mRNA corresponding to these same T7 initiation regions since they also direct the endogenous synthesis of formylmethionine-alanine and formylmethionine-serine. This capability increases as a function of time after infection. In contrast, neither uninfected host ribosomes, ribosomes from T7 am 23 (gene 1 mutant lacking T7 RNA polymerase) infected cells, ribosomes from T7-infected male host cells nor ribosomes from cells infected with Qβ RNA phage direct the synthesis of these or other formylmethionine-dipeptides. It is concluded that the ribosomes after T7 infection seem to have a high affinity for the late viral message and that the regions coding for these formylmethionine-dipeptides may represent preferred sites for their entrance on to the message.

Synthetic DNA (cDNA) complementary to purified mRNA has already proven to be a useful hybridization probe for the quantitation of specific genes and for the detection of specific mRNAs in eukaryotic organisms. In view of its central role in such studies, it seemed necessary to characterize further one such cDNA and to determine the value of established techniques for detecting DNA-RNA hybrids when cDNA is used as a probe. We, therefore, synthesized cDNA complementary to purified rabbit globin mRNA using avian myeloblastosis virus reverse transcriptase. Equilibrium centrifugation in cesium chloride and nearest-neighbor analysis show this cDNA to be heteropolymeric, with a G,C content of 46–48%. Its base composition and nearest-neighbor analysis reflect the expected high frequency of TpT doublets, but also a relatively high proportion of GpG doublets. The cDNA contains at least one T-rich region of sufficient length to form a hybrid with poly (A). Hybridization involving this region is not detected when S i nuclease is used to detect DNA-RNA hybrids. However, this interaction does affect results obtained using hydroxyapatite chromatography. Thus, rabbit globin cDNA exhibits stringent hybridization specificity when assayed using S 1 nuclease, annealing efficiently to rabbit globin mRNA, and not to ribosomal, transfer, or nonglobin mRNAs.

A purified system for protein synthesis, derived from Esherichia coli, makes use of small, synthetic mRNA's initiated by the fMet ‡ codon, AUG, followed by a sequence of 3, 6 or 9 uridylic acid residues. These di-, tri- and tetra-codons direct the binding of fMet- and Phe-tRNA to ribosomes and the synthesis of the corresponding fMet-initiated di-, tri- and tetrapeptides. This system, directing the synthesis of unique and conveniently detected oligopeptide products, has permitted us to correlate the soluble elements required for protein synthesis with specific steps in the translation of initial and succeeding codons. Complete translation of these small mRNA's has certain of the stringent requirements necessary for the accurate cell-free translation of naturally occurring RNA messengers. Thus, initiation factors, fMet-tRNA and GTP must be provided. After recognition of the first codon and formation of the initial complex, one of two transfer factors, in the presence of GTP, catalyzes a binding reaction in which the second (first internal) codon is recognized. This permits formation of the dipeptide, fMet-Phe, but does not permit translation of succeeding codons. A second transfer factor, probably an enzyme, participates in a translocation reaction in which the third codon is made available for translation, apparently displacing the second codon at the recognition site on the ribosome. In addition, the complex formed between peptidyl-tRNA, mRNA and ribosome is stabilized, possibly by translocation of the peptidyl-tRNA from a site of lesser affinity to one of greater affinity on the ribosome. The role of GTP in the translocation reaction will be certain only when it has been uncoupled from its participation in the binding reaction. Once requirements for translation of the third codon have been met no further additions are necessary for elongation of the peptide chain.

A purified preparation of translocase, one of several enzymes required for protein biosynthesis, has been used to prepare a specific anti-translocase antibody. This antibody provides an extremely useful tool not only for detecting the enzyme independent of its activity, but for inhibiting the translocase-mediated reaction and, thus, protein biosynthesis. Though the antibody very rapidly inhibits elongation of the peptide chain, it fails to effect initiation, binding, or peptide bond formation, which strongly suggests that translocase is not a direct participant in these reactions. It also arrests translation of the defined tricodon AUG(U 6 ) at the second codon, permitting formation only of the dipeptide, fMet-Phe, rather than the tripeptide, fMetPhe-(Phe) 2 , which is formed in the presence of control serum. We have shown previously that the third codon becomes available only in the presence of translocase, thereby defining the site of action of the antibody. The antibody also has been used to demonstrate that translocase is antigenically distinct from the other proteins required for initiation and protein synthesis in E. coli .

A cloned line of murine proerythroblastoid cells (T-3-Cl-2), transformed by Friend leukemia virus, undergoes changes associated with erythroid differentiation when treated with dimethylsulfoxide in culture. This line, which does not undergo spontaneous differentiation, develops specific erythrocyte-membrane antigen and accumulates detectable amounts of heme within four days of dimethylsulfoxide treatment. In the present study, we have followed the phenotypic expression of the globin genes by measuring globin mRNA in differentiating cells. Our hybridization probe for this purpose is [ 3 H]DNA, which is complementary to purified globin mRNA, synthesized by viral RNA-directed DNA polymerase. This probe is sufficiently sensitive to detect less than 1 ng of globin mRNA. Using it, we find little or no hybridizable globin mRNA in either uninduced cells or in treated control lymphoid cells. In contrast, globin mRNA can be detected in T-3-Cl-2 cell 2 days after induction by dimethylsulfoxide; it reaches a maximum concentration four days after induction. At this time, cells that stain positively for heme appear. The hybridizable cytoplasmic RNA induced in these cells has the sedimentation properties of 9S globin mRNA. Considering the stable character of globin mRNA, our results are most readily explained in terms of a transcriptional activation of the globin genes.

Small amounts of encephalomyocarditis virus RNA direct a 50-fold increase in amino acid incorporation, in appropriately supplemented ascites tumor cell extracts, under conditions that give rise to authentic viral polypeptides. Incorporation in these crude extracts has a novel characteristic, namely, that it is almost entirely dependent upon the addition of exogenous tRNA. Further, this incorporation is restricted in that tRNA derived from ascites tumor cells or from rat liver permits translation of viral RNA, whereas tRNA from yeast or Escherichia coli does not. These translational barriers are due, at least in part, to an incompatibility between the tRNA of yeast and E. coli and the aminoacyl-tRNA synthetases of the ascites tumor cell. A more extensive basis for this incompatibility is suggested, however, by the failure of the E. coli aminoacyl-tRNA synthetases to restore viral RNA-directed protein synthesis in the presence of tRNA from E. coli , although the coli synthetases fully restore the poly(U)-directed synthesis of polyphenyl-alanine. The possible role that unique or favored codon classes might play in this restriction is considered, together with the implications of the observed requirement for tRNA.

This report describes the properties of a purified Escherichia coli protein synthetic system which accurately translates the initial codons of the bacteriophage f2 maturation, coat, and RNA synthetase cistrons. In order to assay cistron recognition easily and to accumulate specific intermediates in the translation of a natural message, we have restricted in vitro synthesis to the formation of the initial formyldipeptides corresponding to the three f2 cistrons. This is accomplished by preventing the initial, G factor-requiring translocation reaction through the use of small amounts of monospecific anti-G factor antibody or the antibiotic fusidic acid. The reaction requires initiation-competent ribosomes which are depleted of elongation factors by repeated washings in low ionic strength buffer. Initiation in this system is quite specific. The initial f2 coat dipeptide, N-formylmethionylalanine, is readily converted into the expected coat tripeptide, N-formylmethionylalanylserine, if translocation is permitted. In addition, synthesis of the initial f2 RNA synthetase dipeptide, N-formylmethionylserine, is specifically repressed in the presence of f2 coat protein, indicating that this purified system is sensitive to translational control.

Five mutant strains of Escherichia coli were selected for resistance to the steroid antibiotic fusidic acid. The protein synthetic activity of the cell-free extracts of only one of these was resistant to the inhibitory effect in vitro of the antibiotic. G factor, one of the soluble proteins required for peptide chain elongation, was purified to virtual homogeneity from both sensitive parent and resistant mutant strains. Aside from their differing sensitivity to fusidic acid inhibition, the two G factors were indistinguishable by conventional immunological, chromatographic, and electrophoretic analyses. The two factors differed, however, in two other respects. The specific activity of the sensitive strain was approximately twice that of the resistant strain throughout purification. In addition, the Michaelis constant for the ribosomal dependent hydrolysis of GTP calculated for the sensitive factor was half that for the resistant. The study, therefore, characterizes this fusidic acid-resistant mutation as one affecting G factor and establishes a biochemical basis for further genetic studies. It also suggests that the active enzymatic site for the ribosome-dependent hydrolysis of GTP is associated with the soluble G fraction.

RNA derived from encephalomyocarditis virus directs the synthesis of 12 discrete polypeptides in a cell-free system derived from Krebs II ascites tumor cells. The largest of these proteins has a molecular weight greater than 120,000 and accounts for about 40% of the information present in the viral genome. This protein also contains most of the tryptic peptides found in the viral capsid and is analagous to an encephalomyocarditis precursor protein synthesized in vivo. In contrast to the maturation-cleavage mechanism which reduces this large precursor into smaller, mature viral proteins in vivo, the high molecular weight protein does not undergo maturation-cleavage in the cell-free system. Detailed studies involving the pulse-chase technique and peptide analysis of the discrete in vitro products indicate that they arise from a common translational initiation site, but are terminated at several unique points on the viral mRNA.

A rapid, sensitive method is described for measuring C(14)-aminoacyl-sRNA interactions with ribosomes which are specifically induced by the appropriate RNA codewords prior to peptide-bond formation. Properties of the codeword recognition process and the minimum oligonucleotide chain length required to induce such interactions are presented. The trinucleotides, pUpUpU, pApApA, and pCpCpC, but not dinucleotides, specifically direct the binding to ribosomes of phenylalanine-, lysine-, and proline-sRNA, respectively. Since 5'-terminal, 3'-terminal, and internal codewords differ in chemical structure, three corresponding classes of codewords are proposed. The recognition of each class in this system is described. The template efficiency of trinucleotide codewords is modified greatly by terminal phosphate. Triplets with 5'-terminal phosphate are more active as templates than triplets without terminal phosphate. Triplets with 3'- or 3' (2')-terminal phosphate are markedly less active as templates. These findings are discussed in relation to the probable functions of terminal codewords. The modification of RNA and DNA codewords, converting sense into missense or nonsense codewords, is suggested as a possible regulatory mechanism in protein synthesis.

Qbeta is a small bacterial virus whose three genes are encoded in a single-stranded molecule of RNA. This RNA serves directly as the Qbeta message. Here we describe conditions under which RNA corresponding to the coat cistron of this bacterial virus is translated in a system derived from mammalian cells. Translation of the bacterial virus messenger RNA is less effective than that of mammalian globin messenger RNA, but is somewhat enhanced by mild alkali treatment of the messenger. The synthesized product when subjected to electrophoresis migrates with authentic Qbeta coat protein and yields tryptic peptides that correspond to those derived from the Qbeta coat protein.

In order to distinguish among various models which have been advanced to account for the diversity of antibody molecules, we must know how the constant and variable regions of immunoglobulins are represented in the somatic genome. For this purpose, we have purified mRNA corresponding to a mouse kappa immunoglobulin light chain from the myeloma tumor, MOPC-41. This mRNA directs the enzymatic synthesis of highly radioactive DNA (cDNA). This cDNA, which should correspond to the constant region of the kappa chain, was assessed for reiteration frequency using hybridization kinetic analysis and was found to be represented approximately three times per haploid genome . This result tends to rule out germ line hypotheses which require many copies of the constant region gene. It also requires the postulation of a recombinational mechanism to join constant and variable region sequences. Hybridization kinetic analyses designed to assess the entire light chain sequence (C and V regions) made use of 125I-MOPC-41 mRNA. These revealed a major component of relatively unique frequency and a minor (~ 20%) component with a reiteration frequency of approximately 30-50 copies per haploid genome. Careful analysis of the extent of hybridization of this mRNA to DNA prepared from several tumors and tissues, thermal profiles, and relevant competition studies, while sensitive, do not permit us to distinguish unambiguously between a germ line model and the type of somatic mutation model which permits germ line genes corresponding to each kappa subgroup. Our results do, however, clearly rule out the existence of thousands of variable region sequences so closely related to the MOPC-41 V-region as to permit extensive, stable cross hybridization.

During the development of an embryo, various processes occur which induce the formation of differentiated tissues. Some of these processes can be observed at a molecular level, for example, myosin is synthesized in muscle cells, crystaline in lens tissue, keratin in skin, immunoglobulin in lymphocytes and hemoglobin in reticulocytes (1,2).

A cultured, murine erythroleukemic cell line, which initially contains no detectable hemoglobin, can be induced to synthesize hemoglobin in quantities comparable to those found in normal red blood cells. In order to distinguish between several molecular mechanisms which might explain this induction, radioactive DNA complementary to mouse globin messenger RNA was used as a hybridization probe to measure globin genes and mRNA quantitatively during this form of differentiation. The results indicate that the number of globin genes does not change as these cells accumulate hemoglobin and that there are less than five copies of the globin genes per haploid genome. On the other hand, differentiated cells accumulate, on the average, 7000 to 8000 molecules of globin mRNA per cell, compared with less than ten in each undifferentiated cell. The accumulation of globin mRNA ceases in the presence of actinomycin D, suggesting that it is dependent on de novo RNA synthesis. It is also inhibited by cycloheximide and puromycin, suggesting a requirement for continued protein synthesis. Although a mechanism involving the post-transcriptional stabilization of newly synthesized globin mRNA can not be ruled out, these results are most simply explained on the basis of transcriptional activation of globin genes. Further data suggest that the globin mRNA synthesized in these erythroleukemic cells is relatively stable (chemical half-life more than 10 h) and is indistinguishable from authentic reticulocyte globin mRNA. The amount of globin mRNA, synthesized at the rate of approximately 20 nucleotides per second, has been measured and found comparable to the amount in mouse reticulocytes.

Hybridization kinetic analyses with synthetic DNA indicate that there are only two to three copies of the κ constant region gene per haploid genome. This result lends weight to the argument that the immunoglobulin light chain is encoded by more than one continuous gene sequence.

We have purified rabbit globin mRNA using oligo(dT)-cellulose and sucrose gradient centrifugation. Both α- and β-globulin mRNA molecules behave heterogeneously with respect to their elution properties during chromatography on oligo(dT)-cellulose. Those fractions eluted at the lowest ionic strength are most active in directing cell-free globin biosynthesis. By making use of hybridization with synthetic [3H]DNA complementary to globin mRNA, we have shown that this technique can be used to quantitate the extent of mRNA purification. Thus, globin mRNA is approximately 90-fold purified from reticulocyte polysomal RNA and originally constituted slightly more than 1% of the polysomal RNA. Since more than 98% of the globin mRNA sequences are bound to oligo(dT)-cellulose, we suggest that most polysomal globin mRNAs contain a poly (A)-rich region and that this region is not of uniform length nor preponderately associated with either the α- or β-globin mRNAs. In addition, we observe that the 9S globin mRNA most resistant to dissociation from oligo (dT)-cellulose is most active in directing globin biosynthesis.

With oligo(dT)-cellulose as primer, RNA-dependent DNA polymerase catalyzes the synthesis of cellulose-bound DNA that is complementary to mouse globin mRNA. The resulting cellulose-bound or solid phase complementary DNA hybridizes specifically with globin mRNA and permits the recovery of intact globin mRNA. This simple technique for the synthesis of solid phase-bound complementary DNA provides an additional and convenient method for the purification of specific genetic sequences.

Publisher Summary This chapter reviews the molecular events that lead to the translation of mRNA. It discusses the 2-tRNA model for protein synthesis. This system involves the use of an mRNA derived from the small RNA containing coliphage, Qβ. Three general classes of reaction, each producing a discrete ribosomal intermediate, are indicated in the cycle. These are (1) codon recognition, (2) peptide bond formation, and (3) translocation. Each completion of the reaction cycle results in the addition of a single amino acid to the carboxy-terminal end of the growing peptide chain and requires the participation of at least two soluble proteins and a cofactor, guanosine triphosphate (GTP). The chapter discusses the role of each of these proteins, which have by now been purified to virtual homogeneity, and the nature and stoichiometry of GTP utilization. It also discusses the mechanism of the elongation reaction and the mechanism by which mRNA moves across the ribosome—one codon at a time—during protein synthesis.

Certain DNA-containing phage prevent the production of coinfecting RNA phage and shut-off a variety of host functions. It has been suggested that aspects of both these phenomena might be due to specific alterations in the host cell's translational initiation system. In order to explore the role of translational initiation in these processes, we have examined the effect of infection with a variety of phage on the initiation system of Escherichia coli . Our studies viewed cells infected with DNA phage T7 (including a mutant defective in the T7 RNA polymerase) as well as cells infected with the RNA phage Qβ and lysogenic cells induced for the phage λ. Relevant components from infected cells were tested for their ability to carry out the elongation reactions, to synthesize initiation dipeptides corresponding to Qβ and late-expressed T7 cistrons and to recognize the initiation codon, AUG. The most striking effect of infection was a loss of initiation factor activity in all infected cells. In contrast to what might have been expected, the defect induced by T7 infection did not appear to discriminate in favor of translation of late T7 mRNA, but, as with Qβ infection and λ induction, resulted in a general loss of the ability to initiate late T7 and Qβ mRNAs as well as a loss in the ability to recognize the initiation codon AUG. This activity, in the ease of AUG recognition, could be restored to the initiation factors by adding purified IF-1 and IF-2. These results are not easily explained in terms of a model which assumes that phage infection results in altering the specificity of the initiation system.

Nucleotide sequences of RNA codons have been investigated recently by directing the binding of C'4-AA-sRNA to ribosomes with trinucleotides of defined base sequence. The template activities of 12 trinucleotides have been described and nucleotide sequences have been suggested for RNA codons corresponding to phenylalanine, valine, leucine, cysteine, proline, serine, and lysine.l-4 In this report, the template activities of seven additional trinucleotides are described. ApUpU and ApUpC serve as RNA codons for isoleucine, UpApU and UpApC for tyrosine, ApApU and ApApC for asparagine, and ApApG and ApApA for lysine (cf. ref. 1). These findings are discussed in terms of the recognition of specific bases at different positions within a trinucleotide. Materials and Methods.-Components of reactions: E. coli W 3100 ribosomes and sRNA were prepared by modifications of methods described previously.5-7 Each C14-aminoacyl-sRNA was prepared in the presence of 19 C"2-amino acids. The assay for ribosomal bound C14-aminoacyl-sRNA has been described.' Each 50-1 reaction mixture contained 0.10 M1 Tris-acetate, pH 7.2; 0.05 M KCI; 0.03 M magnesium acetate; 1.5-2.0 A260 units of washed E. coli W 3100 ribosomes; and the amount of C14-AA-sRNA indicated in 'Table 1. Other data concerning each C14-AA-sRNA preparation are also given in Table 1. Uniformly labeled C14-amino acids were purchased from New England Nuclear Corp. or Nuclear-Chicago Corp. Radioactivity was determined in a liquid scintillation counter (Packard Inst. Co.) with a C14-counting efficiency of 55-65% as described previously.l Trinucleotides: A derivative of bovine pancreatic ribonuclease was used to synthesize UpApC.8 All other trinucleotides were prepared with a highly purified preparation of polynucleotide phosphorylase.9 The purity of each trinucleotide preparation was assessed as described previously.-4-" 9 The UpApC preparation contained approximately 8% cytidine-2'(3')-phosphate. Other trinucleotide preparatioils appeared to be homogeneous. Analyses of the ApApA preparation have been reported.l The base ratio, base sequence, and chain length of each oligonucleotide were established as shown in Table 2. The methodology employed has been described previously. 4, 9 Results and Discussion.-In Table 3 are shown the effects of eight trinucleotides upon the binding to ribosomes of 17 C-4-AA-sRNA preparations, each acylated with a different C14-amino acid (radioactive Cys-, Met-, and Tryp-sRNA were not used). Each trinucleotide markedly stimulated the binding to ribosomes of only one C14-AA-sRNA preparation. ApUpU and ApUpC stimulated C'4-Ileu-sRNA binding to ribosomes; UpApU and UpApC stimulated C14-Tyr-sRNA binding; ApApC and ApApU stimulated C14-Asp-NH2-sRNA binding; and ApApA and

Before translocation occurs, only the first two codons of each exposed cistron are recognized by the corresponding aminoacyl-tRNAs.

Degenerate serine codons occurring early in the f 2 coat and polymerase cistrons have been used to distinguish between multi-site models of ribosomal function. These studies indicate that only two tRNA's are bound to the ribosome prior to translocation.

Publisher Summary This chapter investigates the enzymatic synthesis of trinucleoside diphosphates of known sequence. Synthesis involves the polynucleotide phosphorylase-catalyzed addition of nucleoside 5′-diphosphate to dinucleoside monophosphate under conditions favoring the production of trinucleoside diphosphate. The products of the reaction are isolated by column chromatography. Trinucleoside diphosphate is the major product, corresponding to a utilization of from 5 to 40% of the dinucleoside monophosphate. Fractions are concentrated and triethylammonium bicarbonate (TEAB) is removed by repeated lyphilization or evaporation to dryness on a rotary evaporator. Oligonucleotides purified in this way are generally contaminated with residual phosphatase activity. Further purification can be achieved by applying approximately 500 A 260 units oligonucleotide per sheet Whatman 3MM paper and developing in descending solvent I for 20 hours. The dinucleoside monophosphate should be applied as a standard. The trinucleoside monophosphate will be the immediately trailing ultraviolet absorbing band. The band may be cut out and eluted from the paper with H 2 O. These compounds are stable at room temperature for 3–6 months when lyphilized and may also be stored frozen in solution for 3–6 months.

In this report, the template activities of twenty-six additional trinucleotides are described and related to the nature of the RNA code. This builds on earlier work of Nirenberg and others, which describes the template activities of nineteen additional trinucleotides and nucleotide sequences suggested for RNA codons corresponding to amino acids. While still far from an "invariant dictionary" at this stage in the research, a table of possible nucleotide sequences is provided.

Bacteriophage Qbeta RNA directs the cell-free synthesis of two fMet-containing dipeptides prior to the first round of ribosomal translocation. One of these, fMet-Ala, corresponds to the initial segment of the Qbeta coat protein. In the present work we take advantage of the fact that translation of the Qbeta RNA polymerase cistron is repressed by its coat protein to correlate the other peptide, fMet-Ser, with the Qbeta RNA polymerase gene. Our experiments show that repression affects translation of the first two codons of the polymerase cistron, thereby isolating the effect of Qbeta coat repressor. Further studies, using single rounds of translocation of the Qbeta mRNA and codon-specific tRNAs, allow us to predict the first three amino acids of the Qbeta RNA polymerase protein, fMet-Ser-Lys, and to suggest that the initiation region of the Qbeta RNA polymerase cistron has the sequence ...AUG.UC[unk].AA[unk]...

Synthetic duck globin DNA has been used to compare the number of genes corresponding to globin in duck reticulocytes and liver cells. The results indicate that the number of globin genes does not differ significantly in these organs and suggest, therefore, that neither amplification nor extensive reiteration of these genes occurs as these two cell types become committed to divergent lines of development.

Publisher Summary This chapter describes an assay for the binding of aminoacyl-transfer RNA (tRNA) to ribosomes. The first step in protein synthesis aminoaeyl-tRNA (AA-tRNA) is bound to ribosomes in the presence of an appropriate oligo- or polyribonucleotide messenger RNA (mRNA) forming a complex reaction. The assay for complex formation involves adsorption of the ribosome and any mRNA directed 14C-AA-tRNA associated with it to a nitrocellulose filter. The Mg++ concentration of Standard buffer is critical. The optimum Mg++ concentration for detecting binding induced by most trinucleotides is 20 mM, whereas it is about 10 mM with polynucleotides. Higher Mg++ concentrations result in increased AA-tRNA binding, which is not dependent upon added mRNA. Ribosomes may be prepared from a number of strains of Escheriehia coli. The low nuclease or nuclease-less strains of Escheriehia coli are particularly useful for the preparation of stable ribosomes. Ribosomes are best stabilized by storing in small aliquots (about 0.1 ml) in liquid N2, freezing and thawing only once. No more than 5.0 A260 of ribosomes should be used in each assay, which is the capacity of the filter. The chapter also discusses the methods of preparing 14C-AA-tRNA.

After the poly-U experiments, the next major challenge was finding out what the remaining codewords were. In this article, the authors use a rapid method for measuring sRNA binding to ribosomes and try to resolve some problems with ambiguous and synonymous codewords. The experimental evidence suggests the binding of specific types of sRNA to ribosomes is directed by only certain corresponding trinucleotides, making sequencing efforts more feasible in the coding process.

ABNORMALLY high concentrations of macroglobulins are known to occur in a number of disease states1. Detection has depended on certain preliminary tests2,3, at times complicated by either false positive or false negative reactions. Definitive evidence is provided only by analytical ultracentrifugation, a procedure not conveniently available at all medical centres.

Several properties of the viral RNA-dependent DNA polymerases and of rabbit globin mRNA make it possible to consider synthesis of the globin gene in vitro . These enzymes copy an RNA template using a short sequence of complementary nucleotides as a primer. Furthermore, globin mRNA has a 3′-terminal sequence of adenylic acid residues that make it particularly suitable as a template, since oligo(dT) can be annealed to a specific site on the mRNA. This small primer could phase the DNA polymerase, possibly ensuring that replication is initiated from that end of the globin message. We have used this approach and find that purified mRNA is an efficient template for the polymerase enzyme. The reaction requires the RNA template and the four deoxyribonucleoside triphosphates, and it is markedly stimulated by the addition of oligo(dT). Consistent with the expectation that the oligo(dT) uniquely phases the polymerase at an adenine-rich region in the globin message, oligo(dG), oligo(dC), and oligo(dA) fail to serve as primers. The product has a density intermediate between that of DNA and RNA, and shifts to a lighter DNA density after treatment with base. Further, it is specifically complementary to globin mRNA and sediments slightly faster in an alkaline sucrose gradient than a DNA standard that has a molecular weight of 129,000. The data suggest that a major portion of the DNA product is a sequence of at least 500 bases, about 50 more than would be necessary to encode rabbit globin. The potential usefulness of this interesting product is discussed.