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Ribonucleic acid and protein synthesis during germination of Myxococcus xanthus myxospores.

Ribonucleic acid (RNA) and protein synthesis during myxospore germination were examined. When RNA synthesis was inhibited more than 90% by either actinomycin D (Act D) or rifampin, germination was prevented. The data were consistent with the interpretation that rifampin did not interfere with protein synthesis in any way other than by inhibition of messenger RNA formation. Act D concentrations as high as 20 mug/ml did not totally inhibit RNA synthesis. In the presence of 8 mug of Act D/ml, germinating myxospores synthesized transfer RNA, 16S RNA, and 23S RNA. Evidence was presented which indicated that messenger RNA was also synthesized early in the germination period both in the presence and absence of 8 mug of Act D/ml. One explanation for the escape synthesis of RNA in germinating myxospores is that Act D exerts a differential effect on the transcription of larger versus smaller cistrons, the latter having a lower probability of binding Act D. We have found that in the presence of 8 mug of Act D/ml, escape RNA synthesis in myxospores was 25% for 23S RNA, 55% for 16S RNA, and more than 90% for 4S RNA. We have shown that germination of myxospores requires both RNA and protein synthesis during the first 25 to 35 min in germination medium. This finding does not support the earlier suggestion by Ramsey and Dworkin that a stable germination messenger RNA is required for germination of the myxospores of Myxococcus xanthus.

Bacteria↗

Temperature-induced derepression of tryptophan biosynthesis in a tryptophanyl-transfer ribonucleic acid synthetase mutant of Bacillus subtilis.

A tryptophanyl-transfer ribonucleic acid (tRNA) synthetase (l-tryptophan: tRNA ligase adenosine monophosphate, EC 6.1.1.2) mutant (trpS1) of Bacillus subtilis is derepressed for enzymes of the tryptophan biosynthetic pathway at temperatures which reduce the growth rate but still allow exponential growth. Derepression of anthranilate synthase in a tryptophan-supplemented medium (50 mug/ml) is maximal at 36 C, and the differential rate of synthesis is 600- to 2,000-fold greater than that of the wild-type strain or trpS1 revertants. A study of the derepression pattern in the mutant and its revertants indicates that the 5-fluorotryptophan recognition site of the tryptophanyl-tRNA synthetase is an integral part of the repression mechanism. Evidence for a second locus, unlinked to the trpS1 locus, which functions in the repression of tryptophan biosynthetic enzymes is presented.

Amino Acyl-tRNA Synthetases↗

Isolation and partial characterization of Escherichia coli mutants with low levels of transfer ribonucleic acid nucleotidyltransferase.

To determine the function of the enzyme transfer ribonucleic acid (tRNA) nucleotidyltransferase in vivo, five mutants of Escherichia coli containing low levels of this enzyme were isolated. Since no selection procedure for such mutants existed, these strains were isolated by assay of large numbers of colonies from a heavily mutagenized stock. A procedure employing cells made permeable to tRNA and ATP was used to screen the large number of colonies required for the isolation. All the mutants contained less than 20% of the normal level of the AMP-incorporating activity of tRNA nucleotidyltransferase in extracts prepared by several methods, and the best mutant contained only about 2% of this activity. Three of the mutants also had equally low levels of the cytidine 5'-monophosphate-incorporating activity of the enzyme. Despite these low activities, the mutant strains displayed relatively normal growth characteristics at all temperatures examined. The enzyme in the mutant strains was not temperature sensitive, nor were any other abnormal biochemical properties detected. tRNA isolated from the mutant strains was missing significant amounts of its 3' terminal adenosine 5'-monophosphate residue, amounting to 10 to 15% in the best mutant. However, only small amounts of the terminal cytidine 5'-monophosphate residue were missing. The results indicate that tRNA nucleotidyltransferase is involved in some aspect of synthesis or repair of the 3' terminus of tRNA, and that the enzyme is present in large excess over its requirements for this function.

Adenosine Monophosphate↗

Effect of arginine on the stability and size of argECBH messenger ribonucleic acid in Escherichia coli.

The chemical stability of argECBH messenger ribonucleic acid (mRNA) produced by Escherichia coli was found to be unaltered during steady-state repression by arginine. During extreme arginine deprivation, the increase in argECBH mRNA stability was related to general effects of amino acid starvation on mRNA stability. Thus a mechanism whereby argECBH gene expression is regulated by altering the decay rate of this mRNA is not consistent with our data. Sucrose gradient analysis followed by hybridization revealed that both heavy (14S) and light (8S) components of argECBH mRNA were produced by cells of E. coli K-12 grown without arginine, whereas predominantly light (8S) mRNA was produced by cells grown with arginine. A functional argR gene and the EC portion of the argECBH cluster were found essential for the arginine restriction of heavy-mRNA production. Experiments suggest that light argECBH mRNA did not arise from heavy message, and 8u% of both light and heavy mRNA was found bound to ribosomes. The data appear most consistent with the notion that a second site of control by arginine regulates the amounts of light and heavy arginine mRNA in the cell either by early termination of transcription or by endonucleolytic processing. Consideration of these data in conjunction with those of the accompanying report (Krzyzek and Rogers, 1976) permits the tentative conclusion that light argECBH mRNA is not translated into active enzymes and is thus responsible for the discrepancy between the high content of hybridizable mRNA and low rates of enzyme synthesis found during arginine repression.

Amidohydrolases↗

Neurospora crassa cytoplasmic ribosomes; ribosomal ribonucleic acid synthesis in the wild type.

The biosynthesis of ribosomal ribonucleic acid (rRNA) In wild-type Neurospora crassa growing at 25 degrees C was investigated by continuous-labeling and pulsechase experiments using [5-3H]uridine. The results of these experiments suggest the following precursor-product relationships: the first RNA molecule to be synthesized in significant quantities is the 2.4 X 10(6)-dalton (2.4-Mdal) ribosomal precursor RNA. This RNA is cleaved to produce two species of RNA with weights of 0.7 and 1.4-Mdal. The former is the mature 17S rRNA of the 37S ribosomal subunit. The 1.4-Mdal RNA is subsequently cleaved to produce the mature 1.27-Mdal (25S) and 61,000-dalton (5.8S) rRNA's of the 60S ribosomal subunit. In the maturation process, approximately 15 to 20% of the 2.4-Mdal ribosomal precursor rRNA molecule is lost. As in other eukaryotes that have been examined, 5S rRNA is not derived from this precursor molecule.

Kinetics↗

New class of ribonucleic acid in Neurospora associated with the outer cell envelope.

Extrinsic ribonucleic acid (RNA) can be isolated from a KCl extract of Neurospora crassa conidial cell surface products. It is heterogeneous in size. The bulk of this RNA travels as a broad band, trailing the 5.8S ribosomal marker RNA on electrophoretic gels. The extrinsic RNA, when denatured, exhibites several discrete lengths between 50,000 and 130,000 daltons. Melting profiles confirm the heterogeneity of the RNA and indicate that 58% of the bases are involved in hydrogen bonding. Analyses of alkaline hydrolysis products reveal no extensive methylation and few or none of the unusual bases present in transfer RNA. The bases are present in approximately equivalent amounts. Extrinsic RNA represents 2 to 3% of the total cellular RNA. Since this membrane-associated class of RNA does not resemble ribosomal RNA, messenger RNA, or transfer RNA and since it is extracted from the cell exterior by methods used to remove extrinsic membrane molecules, we have designated it extrinsic RNA.

Adenine↗

Mode of degradation of precursor-specific ribonucleic acid fragments by Bacillus subtilis.

A precursor of 5S ribosomal ribonucleic acid (rRNA) from Bacillus subtilis was cleaved by ribonuclease (RNase) M5 in cell-free extracts from B. subtilis to yield the mature 5S rRNA plus RNA fragments derived from both termini of the precursor. The released, mature 5S rRNA was stable in these extracts; however, as occurred in vivo, the precursor-specific fragments were rapidly and completely destroyed. Such destruction was not observed in the presence of partially purified RNase M5, so fragment scavenging was not effected by the maturation nuclease itself. The selective destruction of the precursor-specific fragments was shown to occur through a 3'-exonucleolytic process with the release of nucleoside 5'-monophosphates; the responsible activity therefore had the character of RNAse II. Consideration of the primary and probable secondary structures of the precursor-specific fragments and mature 5S rRNA suggested that involvement of 3' termini in tight secondary structure may confer protection against the scavenging activity.

Bacillus subtilis↗

Two modes of metabolic regulation of lysyl-transfer ribonucleic acid synthetase in Escherichia coli K-12.

Lysyl-transfer ribonucleic acid (tRNA) synthetase activity was compared in three independently isolated Escherichia coli K-12 mutants of the enzyme S-adenosyl-L-methionine synthetase (metK mutants) and their isogenic parents. In all three cases the activity of the lysyl-tRNA synthetase was elevated two- to fourfold in the mutant strains. Glycyl-L-leucine (3 mM) usually enhanced lysyl-tRNA synthetase activity two- to threefold in wild-type cells but did not further stimulate the synthetase activity in metK mutants. By two other criteria, the lysyl-tRNA synthetase from wild-type cells grown with the peptide and from the metK mutant RG62, grown in minimal medium, were similar. These criteria are enhanced resistance to thermal inactivation and altered susceptibility to endogenous proteases when compared with the synthetase from wild-type cells grown in minimal medium. In a separate set of experiments, the activities of the lysyl-, arginyl-, seryl-, and valyl-tRNA synthetases were measured in an isogenic pair of relt and rel strains of E. coli grown in a relatively poor growth medium (acetate) and in enriched medium. In the rel+ strain the level of all four synthetases was higher (two- to fourfold) in the enriched medium as expected. In the rel strain the difference in the activities of the synthetases between the two media were diminished. In all four cases the activities of the synthetases were higher in acetate medium in the rel strain. Evidence is presented that these two modes of metabolic regulation act independently.

Amino Acyl-tRNA Synthetases↗

Structure and function of bacteriophage R17 replicative intermediate ribonucleic acid. II. Properties of the parental labeled molecule.

Replicative intermediate ribonucleic acid (RNA), designated RI, which contained parental RNA labeled with (32)P was separated by filtration through agarose from the nucleic acids prepared from (32)P-labeled RNA phage-infected Escherichia coli. A larger amount of ribonuclease-sensitive parental label was found in the rapidly sedimenting forms of RI than in the slower sedimenting forms, indicating that parental RNA is displaced to form a single-stranded tail. This result indicates that some phage RNA is generated by asymmetric semiconservative replication of RI, but it does not mean that a portion of the RI duplexes cannot be conserved during generation of phage RNA. Parental RNA was also found in double-stranded RNA with no apparent tails which sedimented with an S value of 13. This RNA was soluble in 2 m NaCl, and its sedimentation rate was unaffected by ribonuclease; nevertheless, single-strand scissions were produced by ribonuclease and were detected after the duplex was converted to its component single strands.

Coliphages↗

Incorporation of in vitro synthesized reovirus double-stranded ribonucleic acid into virus corelike particles.

When reovirus double-stranded ribonucleic acid (dsRNA) was synthesized in vitro by using a large-particulate fraction (LP-fraction) from reovirus-infected L cells, a significant amount of the (3)H-labeled dsRNA product was incorporated into reovirus corelike particles bound to the LP fraction. These corelike particles were found to be indistinguishable from virus core derived by chymotryptic digestion of virions when compared on the basis of their (i) resistance to chymotryptic digestion, (ii) buoyant density in CsCl, (iii) particle size as determined by agarose chromatography, (iv) elution characteristics from diethylaminoethyl-Sephadex, and (v) resistance of the incorporated (3)H-dsRNA to ribonuclease digestion in 0.01 m NaCl. When the replicase reaction was partially inhibited by NaCl, there was an accumulation of particles that were less dense than the virus core. All of the results indicate that some virus core assembly takes place during the in vitro replicase reaction.

Animals↗

Mechanism of interferon action: inhibition of viral messenger ribonucleic acid translation in L-cell extracts.

Encephalomyocarditis (EMC) virus ribonucleic acid (RNA) stimulated the incorporation of (14)C-amino acids into polypeptides in cell-free systems using preincubated S10 extracts from L cells. Incorporation was linear for over 2 hr. Analysis of the tryptic peptides derived from the polypeptide products formed in response to EMC RNA showed them to be virus specific. The major product, a polypeptide of 140,000 in molecular weight, migrated on sodium dodecyl sulfate-polyacrylamide gels with one of the virus-specific polypeptides present in EMC-infected cells. A minor component of molecular weight about 230,000 may correspond to the product of complete translation of the EMC virus genome. Little or no effect of interferon or vaccinia virus infection was observed in the preincubated, cell-free system. The EMC RNA-stimulated incorporation of (14)C-amino acids into polypeptides was not inhibited in extracts derived from L cells early in virus infection, from interferon-treated cells, or from cells subjected to both treatments. Interferon treatment did appear to have a slight inhibitory effect on chain elongation in this system. However, treatment of cells with highly purified interferon before virus infection caused a decrease of about 80% in the capacity of non-preincubated cell extracts to translate added EMC RNA. This effect did not extend to the translation of polyuridylic acid and could be reversed by preincubation of the extracts at 37 C for 20 min. The inhibition of translation was manifest at interferon concentrations as low as 5IU/ml, and in this respect closely paralleled the inhibition of virus growth. Inactivation of the antiviral activity of the interferon by heating or digestion with trypsin also abolished the effect on cell-free protein synthesis. The EMC-specific polypeptides formed in reduced amounts in extracts of interferon-treated vaccinia-infected cells were smaller than those formed in extracts of untreated, vaccinia-infected cells. Thus, inhibition of initiation or elongation of polypeptides, or both, can be demonstrated in cell-free systems employing non-preincubated extracts from interferon-treated, virus-infected cells. These results indicate that antiviral activity of interferon is directed against the translation of viral messenger RNA.

Amino Acids↗

Measles virus ribonucleic acid and protein synthesis: effects of 6-azauridine and cycloheximide on viral replication.

Cycloheximide and 6-azauridine were employed to study the time course of measles virus protein and nucleic acid syntheses in AV3 cells. Synthesis of ribonucleic acid (RNA) essential for infectivity was first detected at 6 hr and increased concurrently with the formation of essential protein. Maximum levels of virus-specific RNA and protein were present by 18 hr, a time when only 5% of progeny virus was detected. Essential RNA and protein syntheses preceded the formation of infectious virus by at least 10 to 12 hr. The time course of RNA and protein syntheses essential for the formation of complement-fixing (CF) antigen and salt-dependent agglutinin (SDA) was also determined. RNA synthesis essential for the formation of SDA was first detected at 2 hr and was present maximally by 6 hr, whereas SDA-protein increased concurrently with the protein essential for infectivity. This suggested that the last protein essential for infectivity may be SDA. RNA synthesis essential for the formation of CF antigen was first detected at 4 hr, while CF-protein increased at 5 hr and preceded SDA-protein and protein essential for infectivity by approximately 3 hr. Reversal of inhibition of protein synthesis by cycloheximide indicated that early protein synthesis (1 to 3 hr) was required for the formation of infectious virus. The data suggest that the relatively long eclipse period observed with measles virus is related to a long maturation period rather than to late formation of early proteins, viral RNA, or structural proteins.

Amnion↗

Properties of 42S and 26S Sindbis viral ribonucleic acid species.

Two species of ribonuclease-sensitive Sindbis viral ribonucleic acids which sedimented at 42S and 26S were studied. 42S RNA, derived either from virions or from viral nucleoids extracted from infected cultures, was converted by heating to an RNA which sedimented at 26S. The sedimentation patterns of 42S RNA and "derived" 26S RNA were similarly affected in low ionic strength buffers. 42S RNA ran as a homogeneous fraction on polyacrylamide gels; the "derived" 26S RNA as well as "natural" 26S RNA from infected cultures showed similar electrophoretic patterns of heterogeneity. A doubling of 3' polynucleotide termini was observed when 42S RNA was heated. Two possibilities concerning the structure of 42S RNA are considered. (i) It may consist of an aggregate of subunits, joined by means of hydrogen bonds to form a complex molecule. (ii) A heat-labile covalent bond of unknown type may link viral RNA subunits. Although 26S RNA from infected cultures and "derived" 26S RNA from 42S RNA behaved in a similar qualitative manner on gels, their sedimentation characteristics were affected differently in low ionic strength buffers. "Natural" and "derived" 26S RNA appear to consist of a population of fragments. and their behavior in gradients and in gels is probably dictated by the experimental conditions of the analytical methods used.

Animals↗

Ribonucleic acid polymerase induced in cells infected with Sendai virus.

A ribonucleic acid (RNA)-dependent RNA polymerase was induced in chick embryo fibroblast cells after infection with Sendai virus (parainfluenza 1 virus). The enzyme was associated with the microsomal fraction of infected cells and reached maximum detectable activity at 18 hr after virus infection. The activity of the enzyme in vitro was dependent on the presence of added magnesium ions and all four nucleoside triphosphates and was not inhibited by actinomycin D. The RNA synthesized by the enzyme in vitro was sensitive to ribonuclease and consisted of a complex mixture of RNA species including 34S, 24S, and 18S components. Similar RNA components were detected in the microsomal fraction of Sendai virus-infected cells by labeling with (3)H-uridine from 17 to 18 hr postinfection in the presence of actinomycin D. Of the RNA synthesized by Sendai virus-induced RNA polymerase in vitro, 98% became insensitive to ribonuclease after annealing with RNA extracted from purified Sendai virus particles.

Animals↗

Saint Louis encephalitis viral ribonucleic acid replication complex.

Pulse-labeled Saint Louis encephalitis viral ribonucleic acid (RNA) is found in the cytoplasm of infected cells associated with a membranous structure which sediments with an average value of 250S. The integrity of the complex is destroyed by detergents and ribonuclease; however, it is stable in ethylenediaminetetraacetic acid (EDTA) which differentiates this structure from cellular polyribosomes. With cultures in which cellular RNA was highly labeled prior to infection, ribosomal RNA could not be demonstrated in the complex isolated from EDTA-sucrose gradients. Single-stranded 43S and the 26S and 20S forms of viral RNA were found in the complex. Viral RNA polymerase activity in sucrose-gradient fractions sedimented in the same region as the fractions which contained the pulse-labeled viral RNA. The polymerase incorporated (3)H-guanosine triphosphate into acid-precipitable material in the absence of added template. It was also found that the replication complex contains viral-specific proteins.

Animals↗

Expression of insulin-like growth factor (IGF), IGF-binding protein, and IGF receptor messenger ribonucleic acids in normal and polycystic ovaries.

Expression of the messenger ribonucleic acids (mRNAs) for insulin-like growth factors (IGFs), their binding proteins (IGFBP1 through IGFBP-6), and receptors was examined in normal and polycystic human ovaries (PCO). Northern and dot blots and RT-PCR were used to evaluate mRNA levels. The IGF system was studied in fresh granulosa, stromal, and thecal samples and in thecal tissue after incubation with LH and GH. IGF-II expression was high in granulosa and thecal compartments, whereas IGF-I was only weakly detectable, suggesting the importance of IGF-II in the human ovarian IGF system. Northern blot analysis revealed IGFBP-2 and -4 mRNA in all ovarian compartments and IGFBP-5 mRNA in stroma and theca. IGFBP-2 mRNA was the most abundant IGFBP mRNA in the human ovary. No IGFBP-1 or -3 mRNA was detected in fresh ovarian tissues. IGFBP-6 and type 1 and 2 IGF receptor mRNA expression was detectable in all ovarian compartments only by RT-PCR. In cultured theca, the expression of IG-FBP-1, -3, and 4 was induced. Only IGFBP-5 expression showed some dependence on trophic hormone (LH) stimulation during theca incubation. Otherwise, GH and LH had no effect on IGF or IGFBP expression in thecal tissue. This study indicates that thecal tissue is an essential part of the IGF system in the human ovary. However, no differences were found between normal ovaries and PCO in IGF, IGFBP, or IGF receptor expression. Thus, our results (from a limited number of patients) together with recent in situ hybridization and immunohistochemistry data of others provide no evidence for a role for IGFs in the functional disturbances related to PCO. Interestingly, our data show that in cultured thecal samples, the IGF and IGFBP expression pattern is different from that in fresh tissue.

Adult↗

The development of fixed-ratio performance under the influence of ribonucleic acid.

The transition from fixed-ratio 1 performance (every response reinforced) to fixed-ratio 30 performance (every thirtieth response reinforced) was studied in nine pigeons. These were divided into three treatment groups given daily oral doses of saline, or 250 mg/kg/day or 500 mg/kg/day of yeast ribonucleic acid. Detailed computer-assisted analyses of how fixed-ratio behavior develops revealed the following typical sequence. After the transition, the first few ratios typically were emitted without long interresponse times within the ratio. Steady responding then ceased, and numerous long interresponse times occurred, with no systematic relationship to ordinal position within the ratio. Gradually, a new pattern evolved, characterized by a consistently long post-reinforcement time, a border region of the next few interresponse times within which the mean interresponse time monotonically decreased, and short interresponse times within the last 80% of the ratio. Long interresponse times were eliminated from this last section of the ratio without regard to proximity to reinforcement. Various analytical procedures suggested that the final pattern can be conceived, in part, as the shaping of a reliable response topography. The group of three pigeons given 250 mg/kg/day of yeast ribonucleic acid responded at higher rates than the saline and 500 mg/kg/day groups. The latter group, in contrast to the saline and lower dose groups, which continued to increase their rates, reached a rate asymptote very early.

Administration, Oral↗

Platelet-derived growth factor and double-stranded ribonucleic acids stimulate expression of the same genes in 3T3 cells.

Platelet-derived growth factor (PDGF) stimulates expression of a "competence" gene family in Balb/c-3T3 cells. The competence family contains the c-myc and c-fos genes together with several functionally uncharacterized genes (JE, KC, and r-fos) that have been isolated as cDNA clones. We show that double-stranded ribonucleic acid is a potent inducer of the competence gene family. Infection with vesicular stomatitis virus also induces expression of this gene family. Conversely, PDGF stimulates expression of genes hitherto characterized as responsive to double-stranded ribonucleic acids, including the beta-fibroblast interferon and (2'-5')-oligoadenylate synthetase genes. These PDGF-inducible genes could conceivably function in a feedback loop to control 3T3 cell growth. Some of the genes, such as c-fos and c-myc, are induced quickly by PDGF and may initiate a round of cell division. Others, such as beta-fibroblast interferon and (2'-5')-oligoadenylate synthetase, are induced more slowly and may function as feedback inhibitors of the growth response to PDGF.

2',5'-Oligoadenylate Synthetase↗