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Sm and Sm-like proteins are members of a family of small proteins that is widespread throughout eukaryotic kingdoms. These proteins form heteromers with one another and bind, as heteromeric complexes, to various RNAs, recognizing primarily short U-rich stretches. Interestingly, completion of several genome projects revealed that archaea also contain genes that may encode Sm-like proteins. Herein, we studied the properties of one Sm-like protein derived from the archaebacterium Archaeoglobus fulgidus and overexpressed in Escherichia coli . This single small protein closely reflects the properties of an Sm or Sm-like protein heteromer. It binds to RNA with a high specificity for oligo(U), and assembles onto the RNA to form a complex that exhibits, as judged by electron microscopy, a ring-like structure similar to the ones observed with the Sm core ribonucleoprotein and the like Sm (LSm) protein heteromer. Importantly, multivariate statistical analysis of negative-stain electron-microscopic images revealed a sevenfold symmetry for the observed ring structure, indicating that the proteins form a homoheptamer. These results support the structural model of the Sm proteins derived from crystallographic studies on Sm heterodimers and demonstrate that the Sm protein family evolved from a single ancestor that was present before the eukaryotic and archaeal kingdoms separated.

Erwinia herbicola, a nonphotosynthetic bacterium, is yellow colored due to the accumulation of unusually polar carotenoids, primarily mono- and diglucosides of zeaxanthin. We have cloned and expressed the gene for the enzyme that catalyzes the glucosylation of zeaxanthin. The enzyme has an apparent molecular mass of 45 kDa on an SDS/polyacrylamide gel, which is consistent with its calculated molecular mass. In vitro enzymatic activity was demonstrated using UDP-[14C]glucose and zeaxanthin as substrates. The product zeaxanthin diglucoside and its intermediate monoglucoside were identified by thin layer chromatography. The optimum pH and temperature ranges of the enzyme are 7.0-7.5 and 32-37 degrees C, respectively. A hydropathy plot indicates no apparent membrane-spanning regions, and biochemical experiments suggest that the enzyme is weakly membrane-associated. The amino acid sequence derived from the zeaxanthin glucosyltransferase gene shows a small region of high similarity with other glucuronosyl- and glucosyltransferases that use either UDP-activated glucuronic acid or a sugar as one of their substrates. Based on these similarities, we propose that this conserved sequence is part of the UDP binding site.

Cyanogen bromide treatment of thymidylate synthetase of Lactobacillus casei, which had been converted to a ternary complex with [2-14c] FdUMP and 5,10-methylene-tetrahydrofolate followed by S-carboxymethylation, yielded at least four visible peptide bands, the largest with a molecular weight of about 13,000, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate-urea. Identical results were obtained with enzyme that had all four of its cysteinyl residues S-carboxymethylated with iodo [I-14C] acetate in the absence of FdUMP and cofactor. In each case, only the second band from the top of the gel (CN2), with an approximate molecular weight of 10,000= was labeled. Analysis of CN2 that had been labeled with [2-14C] FdUMP and nonradioactive iodoacetate and of that labeled only with iodo[1-14C] acetate revealed that their amino-acid contents were almost identical except for the presence of two S-carboxymethyl (Cm)-cysteinyl residues in the latter peptide and only one in FdUMP-CN2. A nonapeptide was isolated from (Cm)2-CN2 after chymotrypsin digestion that contained the following sequence by dansyl-Edman analysis: Ala-Leu-Pro-Pro-[Cm-Cys]-His-Thr-Leu-Tyr. This peptide was found to be located on the NH2-terminal end of CN2. Automatic sequence analysis of the first 13 residues of (Cm)2-CN2 and of the FdUMP-containing CN2 yielded identical results except for the fifth, or cysteinyl, residue, which could not be identified in the latter peptide. These findings strongly suggest that FdUMP is linked to a cysteinyl residue in thymidylate synthetase that has been inactivated irreversibly by this nucleotide.

Three classes of heterogeneous nuclear RNA (HnRNA) alpha, beta, and gamma, with different chemical and physical properties, can be identified in the early sea urchin embryo by hybridization with poly(U). The relative amounts of these classes vary as a function of embryonic development. It is demonstrated here that the adenyl-containing classes of HnRNA, alpha and beta, can be subfractionated by hybridization with poly(A)-agarose into species containing and lacking oligo(U)-enriched segments. These oligo(U) segments could not be detected in gamma HnRNA, which was previously shown to also lack adenylate segments. The relative proportions of these species undergo marked changes during development from early blastula (7 hr) to mesenchyme blastula (20 hr). I propose models to explain the possible effects of complementary sequences of adenylate and uridylate on the secondary structure of HnRNA, and speculate on the functional significance of such complexes.

The effects of the src gene on the activity of protein kinase C and intercellular communication have been studied in transformed NIH/3T3 clones isolated from soft agar following transfection with the plasmid carrying the v-src gene (psrc-11). Six transformed clones that were studied contained newly incorporated v-src genes in the genome, had an increased amount of pp60src, and showed enhanced activities of protein kinase C. Intercellular communication, studied by observing with autoradiography the transfer of [3H]uridine nucleotide from prelabeled donor cells to recipient cells in contact, was found to be reduced in transformed clones as compared to parental NIH/3T3 cells. Treatment with phorbol 12-myristate 13-acetate was also found to increase protein kinase C activity and to reduce intercellular communication in normal NIH/3T3 cells. These results suggest that the v-src gene product, in a manner similar to some of the powerful tumor promoters, may directly or indirectly affect cell-cell communication.

Purified beta-1,4-N-acetylmuramoylhydrolase (muramidase-1; EC 3.2.1.17) of Streptococcus faecium ATCC 9790 has been shown to be covalently substituted with approximately 12 mol equivalents of monomeric 5-mercaptouridine monophosphate. All 12 residues are present on the proteolytically processed 87-kDa active form of the enzyme. A peptide fragment containing 5-mercaptouridine, tyrosine, alanine, glycine, and leucine was isolated consistent with an O-phosphate linkage of the nucleotide to tyrosine.

Glutamine-dependent carbanoyl phosphate synthase [ATP6carbamate phosphotransgerase (dephosphorylating), EC 2.7.2.9], aspartate transcarbamoylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) and dihydroorotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3), are copurified as a high-molicular-weight complex from extracts of unfertilized eggs of Rana catesbeiana. UTP is required to maintain the integrity of the complex during the last two purification steps. Removal of the nucleotide results in dissociation of the complex. Based on sedimentation behavior in glycerol gradients, the dissociated carbamoyl phosphate synthase has an apparent molecular weight of 260,000 +/- 20,000 and that of dihydroorotase is estimated at 280,000 +/- 20,000. Aspartate transcarbamoylase is broadly distributed over the gradient. The addition of ATP, 5-phosphoribosyl-1-pyrophosphate, Mg++, or inorganic phosphate to the dossociated complex results in the appearance of a peak of aspartate transcarbamoylase activity with an apparent molecular weight of 110,000 +/- 10,000. Icubation of a mixture of the dissociated enzymes with UTP and Mg++ leads to their reassociation into the high-molecular-weight complex.

Nuclear monolayers of WI-38 cells prepared by the method of Tsai and Green were used to determine RNA synthesis in isolated nuclei in situ . In nuclear monolayers, incorporation of [ 3 H]UTP into RNA is dependent on the presence of the other three nucleotide triphosphate and is abolished by actinomycin D. The extent of RNA synthesis under these conditions was measured in density-inhibited WI-38 human diploid fibroblasts at various intervals after cell proliferation was stimulated by a change of medium. RNA synthesis increases 15 min after the nutritional change and reaches a peak at 18 hr, which is also the peak of DNA synthesis. Thereafter RNA synthesis declines. Essentially similar results are obtained whether the endogenous RNA polymerase or a bacterial polymerase is used. Replacement of the stimulating medium by conditioned medium stops the increase in RNA synthesis that occurs in cultures subject to continuous stimulation. Finally, RNA synthesis in nuclear monolayers, using the endogenous RNA polymerase, occurs by chain elongation only, while re-initiation occurs with the bacterial RNA polymerase.

Platinum binding to nucleoside phosphorothioates has been examined to determine their suitability as heavy metal labeling sites for the potential electron microscopic sequencing of nucleic acids. The complex platinum terpyridine nitrate forms a 1:1 adduct with either adenosine or uridine monophosphorothioate. Spectroscopic evidence strongly indicates the presence of platinum-sulfur bonds. Both platinum terpyridine nitrate and chloroterpyridineplatinum(II) bind to poly(sA-U), a polymer prepared from adenosine 5'-O-(1-thiotriphosphate) and UTP. Binding to the sulfur atoms of the phosphorothioate groups is quantitative, as shown by double label experiments using [35S]poly(sA-U) and [3H]chloroterpyridine-platinum(II). Similar experiments with [14C]poly(A-U) indicated no platinum binding. No evidence of nicking or loss of sulfur from poly(sA-U) could be detected after platinum binding. The phosphorothioate group is a strong, highly selective binding site for platinum in polynucleotides. Previous studies have demonstrated quantitative enzymatic incorporation of phosphorothioate groups into a polynucleotide adjacent to a specific base [Matzura, H. & Eckstein, F. (1968) Eur. J. Biochem. 3, 448-452]. The use of heavy metal-labeled phosphorothioate groups for the sequencing of nucleic acids by electron microscopy therefore appears feasible.

The relation between cellular uracil nucleotides and ability to synthesize glycogen was studied in rat diaphragm incubated in vitro. In the absence of exogenous uridine the tissue content of UTP and rate of glycogen synthesis decreased with time. Uridine added to the medium increased cellular UTP and UDPG and stimulated glycogen synthesis. Insulin significantly increased the synthesis of UTP from extracellular uridine. This action of insulin appeared to be due to a stimulation of phosphorylation of the nucleoside and not to an effect on transport at the concentrations of uridine studied. However, an effect of insulin on transport of uridine at low concentrations cannot be excluded.

Advantage was taken of the reversibility of the first step of ribonuclease-A action to synthesize the dinucleoside phosphorothioate Up(S)C from the crystalline isomer of uridine 2′,3′-cyclic phosphorothioate [Up̂(S)] and cytidine. Cyclic phosphorothioate was then reformed from Up(S)C by a nonenzymic reaction known to proceed by an in-line mechanism. The geometry of the enzymic reaction was determined to be in-line by a comparison of the Up̂(S) product with the Up̂(S) originally used. By the principle of microscopic reversibility, the geometry of the first step in the action of ribonuclease-A is shown to be in-line.

Four peaks of DNA-directed RNA polymerase activity are resolved by salt gradient elution of a sonicated yeast cell extract on DEAE-Sephadex. The enzymes, which are named IA, IB, II, and III in order of elution, all appear to come from cell nuclei. Only enzyme II is sensitive to α-amanitin. All enzymes are more active with Mn ++ than with Mg ++ as divalent ion. Enzymes IB and II have salt optima in the range 0.05-0.10 M (NH 4 ) 2 SO 4 , whereas enzyme III is maximally active at 0.20-0.25 M (NH 4 ) 2 SO 4 . With optimal salt concentration and saturating DNA, the template preference ratio, activity on native calfthymus DNA divided by activity on denatured calf-thymus DNA, is 2.2 for IB, 0.4 for II, and 3.5 for III. None of the yeast polymerases was inhibited by rifamycin SV. Rifamycin AF/013 effectively inhibited polymerases IB, II, and III.

The specific activity of mouse neuroblastoma choline- O -acetyltransferase (EC 2.3.1.6.) increased 5.7-fold when the rate of cell division was restricted (as compared to cells kept rapidly dividing for 9 days); the specific activity of mouse neuroblastoma thymidylate synthetase increased 2.4-fold when nondividing cells again entered the logarithmic phase of cell growth. The highest specific activities for choline- O -acetyltransferase and lowest specific activities for thymidylate synthetase were obtained from cultures where cell division was restricted; the opposite result was observed when the cells were growing rapidly. Thus, the regulation of these two enzymes is out of phase with respect to each other and is dependent on the rate of cell division. The inverse relationship for the regulation of these two enzymes is discussed in relation to the needs of mitotic versus differentiated neuroblastoma cells.

Behavior of synthetic polyribonucleotides in cell- free amino-acid-incorporating systems from E coli, noting release of polypeptide chains into supernatant fraction

Fluorescence of quinacrine in the presence of different polynucleotides was studied to attempt to identify the specific nucleotides responsible for the fluorescence of stained chromosome preparations. A marked enhancement of fluorescence was seen in the presence of bihelical polynucleotides, such as poly(dA-dT), poly(dA)·poly(dT), and poly(rA)·poly(rU), but not in the presence of single-stranded polynucleotides, such as poly(dA), poly(dT), poly(rA), or poly(rU) alone. The higher was the GC content of natural DNAs, the more they quenched. Quenching was also seen with poly(dG) or poly(rG) alone, but not with poly(dC) or poly(rC) alone. Native and denatured DNA were both effective in quenching fluorescence. Thus, a bihelical conformation is not required for fluorescence quenching. Nearly all of these properties are shared with proflavine. In contrast, acridine orange, which stains most areas of chromosome preparations, shows enhanced fluorescence in the presence of all members of a series of natural DNAs. These data suggest that base-pairs composed of AT (rather than GC) residues are responsible for the observed fluorescence of specific chromosome regions after treatment with quinacrine, and support the proposal of Ellison and Barr ( Chromosoma , in press) that the highly localized quinacrine fluorescence in their cytological preparations reflects the presence of DNA that has a high (A + T)/(G + C) ratio.

The antiviral activity and interferon-inducing ability of single-, double-, and triple-stranded polynucleotides, modified at pyrimidine C-5 or purine N-7, were evaluated in primary rabbit kidney cells challenged with vesicular stomatitis virus. ( 1 ) There is a parallel increase in antiviral activity and the temperature at which double-stranded polynucleotides rearrange to inactive triple-stranded complexes. ( 2 ) When the purine N-7 of (A) n is replaced by CH, all resulting double-stranded complexes fail to provide antiviral protection or to induce interferon, even though such complexes meet all requirements previously recognized for interferon induction. ( 3 ) Competition experiments between inactive and active polynucleotides indicate that single-stranded polynucleotides apparently do not bind to the cellular receptor sites for interferon induction, whereas triple-stranded complexes and inactive double-stranded complexes bind to such receptor sites but, probably for conformational reasons, fail to trigger the necessary message for interferon induction.

During in vitro incubation, nuclei from polyoma-infected cells elongate the daughter strands of the replicative intermediate of polyoma DNA. This process is now shown to involve the transient formation of short fragments (4-5 S), a process that is stimulated by the addition of ribonucleoside triphosphates. The presence of stretches of RNA at the 5′-end of short DNA chains was determined from Cs 2 SO 4 equilibrium centrifugation and from the finding that isotope from α- 32 P-labeled deoxynucleoside triphosphates was recovered in 2′(3′)-ribonucleotides after alkaline hydrolysis. Transfer occurred preferentially with [α- 32 P]dCTP as substrate. Starvation for deoxynucleotides by in vivo treatment with hydroxyurea resulted in the accumulation of short fragments that are deficient in RNA. Our results suggest that a late step during the discontinuous synthesis of polyoma DNA is selectively inhibited when deoxynucleotides are in short supply.

Mitochondrial RNA polymerase activity from rat liver has previously been demonstrated in intact organelles. This activity has now been solubilized, partially purified, and shown to be a true polymerase, free of nuclease. The enzyme is derived from mitochondria and is not from contaminating bacteria or nuclear components. The enzyme is distinguished from its nuclear counterparts by its behavior on ammonium sulfate fractionation and lack of inhibition by α-amanitin. Rifamycin inhibits the crude enzyme, but only inconsistently inhibits the more purified preparation.

Negative cooperativity has been observed in CTP synthetase, an allosteric enzyme which contains a regulatory site. Thus, the same enzyme exhibits negative cooperativity for GTP (an effector) and glutamine (a substrate) and positive cooperativity for ATP and UTP (both substrates). In the process of the delineation of these phenomena, diagnostic procedures for negative cooperativity were developed. Application of these procedures to other enzymes indicates that negative cooperativity is a characteristic of many of them. These findings add strong support for the sequential model of subunit interactions which postulates that ligand-induced conformational changes are responsible for regulatory and cooperative phenomena in enzymes.

those of more complex organisms; (2) the lack of growth and of extensive qualitative changes at the enzyme level; and (3) the availability of the various kinds of information required to fully analyze the "fine-level" control of metabolism: cellular levels of substrates, products, coenzymes, and effectors; enzyme specific activities, affinity constants, etc.; and most importantly, the rates in vivo of critical reactions necessary to differentiation. The data that have now accumulated relevant to the intermediary metabolism of the cellular slime mold have reached a stage of complexity requiring a quantitative dynamic model for further analysis. The range of possible control mechanisms and their interdependence have become so complicated that it is difficult to distinguish cause from effect and random variation from significant change.' The kinetic model to be presented is preliminary and must become more complex and sophisticated as other factors known to be involved are included and as new data become available. However, even in its present simplified form, the model has provided a valuable conceptual framework for thinking in kinetic terms and for understanding some of the complexities of the differentiating cell; it has revealed new relationships and objectives not previously apparent and has made possible specific predictions, which are now being tested in the laboratory. Data Relevant to the Model.-Differentiation in the cellular slime mold can be described as a "closed system" in the sense that the cells are starving and independent of exogenous nutrients; dry weight decreases to about half the original value. Total carbohydrate content, based on the original dry weight, remains fairly constant throughout development.2-4 By the end of the differentiation process, it is possible to account very roughly for the total carbohydrate (anthrone-positive) material (about 14% of the final dry weight) in terms of identifiable compounds. Expressed in glucose equivalents/ml of packed cells (--100 mg dry weight) and as per cent of the original dry weight, these materials

When UDP-galactose 4-epimerase (EC 5.1.3.2) from Escherichia coli is incubated with UDP-galactose, then reduced with NaB 3 H 4 , label is found in UDP-glucose and UDP-galactose. Enzymatic and chemical degradation demonstrates that the label is bound to carbon 4 of the glycosyl moieties. These results provide direct evidence for the existence of UDP-4-keto-glucose as an enzyme-bound intermediate in the epimerization reaction, and they exclude the formation of a 3-keto intermediate.

The effects of dinucleoside monophosphates on the transcription of phage T4 DNA by E. coli RNA polymerase have been examined at various concentrations of the sigma subunit and extremely low concentration of ribonucleoside triphosphate. The following conclusions were reached: ( i ) Labeled specific dinucleoside monophosphates are incorporated as chain initiators. ( ii ) When the ratio of sigma factor to core enzyme is small, there is a general stimulation by most 5′-guanosyl dinucleoside monophosphates. ( iii ) When the ratio is increased or holoenzyme is present, ApU, CpA, UpA, and GpU are the most effective stimulators. ( iv ) At high concentrations of sigma factor, only certain adenosine-containing dinucleoside monophosphates (ApU, CpA, UpA, and ApA) stimulate the reaction. ( v ) Competition hybridization studies indicate that the RNAs stimulated by dinucleoside monophosphates (ApU, CpA, UpA, and GpU) are of the T4 “early” type. ( vi ) Studies involving both combinations of stimulatory dinucleoside monophosphates and competitive effects of these compounds on chain initiation by ATP and GTP suggest that the stimulatory dinucleoside monophosphates act as chain initiators and may recognize part of a continuous sequence in a promoter region. Studies based on the incorporation of 3 H-labeled stimulatory dinucleoside monophosphates support the above conclusions.

Intraventricular administration of dibutyryl adenosine cyclic 3′:5′-monophosphate into goldfish brains produced hyperactive animals. A study of the effects of the drug (25-50 mg/kg) on the incorporation of [5- 3 H] orotic acid, as a precursor of labeled uridine and cytidine, into newly synthesized RNA showed the formation of an RNA with a uridine to cytidine ratio 20-50% higher than that of the control. In double-labeling experiments with uridine as the labeled precursor, the synthesis of a nuclear RNA fraction (not produced in the absence of drug) was demonstrated. Some of this RNA was found to migrate into the cytoplasmic fraction and to become associated with polysomes. The results suggest that cyclic AMP might function as a “metabolic demand signal” for eliciting new RNA synthesis in goldfish brain.

In an attempt to ascertain whether specific base-pairing could have a regulating function in an enzyme system in which nucleotides serve as the substrate, we studied the effects of the addition of the various homopolymers of the ribonucleotide series on the action of adenylate kinase on ADP or CDP, and found that the results support this possibility. Poly U strongly inhibits the enzyme action on ADP, and poly G that on CDP, both to the extent of about 70 per cent. Lesser effects are shown when poly G is used with ADP and poly U with CDP. The other homopolymers tested, poly A and poly C, are ineffective.

Earlier studies showed that two protein components, P I and P II , are concerned with the adenylylation and deadenylylation of Escherichia coli glutamine synthetase (EC 6.3.1.2). P I by itself catalyzes both adenylylation and deadenylylation, but its activity is modulated by the P II -protein and by glutamine, 2-oxoglutarate, ATP, and UTP, The P II -protein exists in two forms: one form, P II -AT, stimulates P I -catalyzed adenylylation activity in the absence of glutamine and makes this activity very sensitive to inhibition by 2-oxoglutarate; it does not affect deadenylylation activity. The other form, P II -DA, stimulates adenylylation only if glutamine is present, and also stimulates the deadenylylation activity of P I , which is then dependent upon the presence of ATP and 2-oxoglutarate. Conversion of P II -AT to P II -DA requires the presence of UTP, ATP, and 2-oxoglutarate; it is catalyzed by an enzyme present in P I preparations. UTP may be directly involved in this conversion since P II -DA fractions reisolated by filtration through Sephadex G-100 contain small quantities of a bound uridine derivative that lacks the γ-phosphoryl group of UTP. The activity of P II -DA, but not of P II -AT, is destroyed by treatment with snake-venom phosphodiesterase. ATP and 2-oxoglutarate apparently function as allosteric effectors for the conversion of P II -AT to P I -DA.

Evidence for hydrogen bonding between 5′-ribonucleotides in water has been obtained from a 220-MHz proton magnetic resonance study of nitrogenous protons. The amino groups of GMP, AMP, and CMP exhibit proton resonance lines which are somewhat broadened by proton exchange with the solvent at 0°; their downfield Shifts in mixtures of mononucleotides provide the basis for the following order of base-pairing tendencies: GMP·CMP > AMP·UMP. Hydrogen bonding is also observed in other pairs of mononucleotides, notably GMP·UMP, AMP·CMP, and CMP·UMP, to a lesser extent in GMP·IMP, CMP·XMP, and possibly in CMP·IMP. In agreement with previous reports, hydrophobic interactions of mononucleotides have also been observed; base pairing occurs in addition to vertical stacking of these bases, their hydrogen bonding to water, or self-association. Only CMP shows clear evidence of self-association via hydrogen bonding in water; the evidence for GMP is less direct, and that for AMP is negative. This lack of observable self-association may occur as a result of competition from strong stacking interactions. Only CMP shows restricted rotation of the amino group at 0° and neutral pH. As expected, higher temperatures increase the rate of rotation of the amino group for CMP, as well as accelerate the rate of proton exchange between water and the amino protons of mononucleotides. High-resolution proton magnetic resonance spectroscopy could prove to be a valuable tool in mapping out the specificities conferred by hydrogen bonding between biomolecules in aqueous solution.

T4 polynucleotide ligase efficiently catalyzes the head-to-tail joining of the ribo-oligoadenylates, r-(pA) 8 and r-(pA) 10 , in the presence of high molecular weight deoxypolythymidylate. The enzyme also catalyzes the joining of deoxy-oligothymidylates, e.g., d-(pT) 10 , in the presence of ribopolyadenylate. The enzyme failed to bring about the joining of r-(pA) 10 when poly r-U was used as the template, although a slow formation of the expected activated intermediate from r-(pA) 10 was detected.