Pulse-labeled Ribonucleic Acid Research Articles

Relationship Between Messenger Ribonucleic Acid and Enzyme Levels Specified by the Leucine Operon of Escherichia coli K-12

The levels of leucine-forming enzymes in Escherichia coli K-12 varied over a several thousand-fold range, depending upon conditions of growth. The highest levels were achieved by growing auxotrophs in a chemostat under conditions of leucine limitation. Under such conditions, enzyme levels were increased 45- to 90-fold relative to cells grown in minimal medium containing leucine (the latter values arbitrarily called 1). Leucine operon-specific messenger ribonucleic acid levels were elevated to about the same extent as enzyme levels in cells grown in a chemostat. Growth in media of greater complexity resulted in progressively lower levels of leucine-forming enzymes, reaching a value of less than 0.02 for growth in a medium containing tryptone broth and yeast extract. The levels of leucine operon-specified enzymes and messenger ribonucleic acid were also measured in strains containing about 25 copies of plasmid pCV1(ColE1-leu) per chromosome. For such strains grown in minimal medium, enzyme levels were proportional to the number of plasmids per cell. Furthermore, they followed the same trends as those described above upon derepression in a chemostat or upon repression following growth in rich media. Leucine messenger ribonucleic acid, measured both by pulse-labeling and hybridization-competition experiments, was roughly proportional to enzyme levels over this entire range. For a plasmid-containing strain grown in a chemostat under conditions of leucine limitation (about 100 plasmids per chromosome), about 27% of pulse-labeled ribonucleic acid was coded for by genes in or adjacent to the leucine operon, and 10% of the total protein was beta-isopropylmalate dehydrogenase.

Characteristics of pulse-labeled ribonucleic acid in relation to medium pH and acetate concentration during meiosis of yeast

Cellular impermeability associated with sporulating cells of Saccharomyces cerevisiae is caused by a rapid increase in the medium pH. Three factors have been identified as being important in regulating the rise in medium pH: 1) the cell density, 2) the potassium acetate concentration of the sporulation medium, and 3) and initial pH below 6.0. Sporulation conditions were established for strain 4579 which resulted in optimum uptake of 3H-adenine at T7, a period when the cells would be normally impermeable. Pulse-labeled polysomal RNA was characterized at T4 in naturally permeable cells of strain SK-1 and impermeable cells which required manipulation of the medium pH to facilitate uptake. Transfer ribonucleic acid (RNA), poly A-containing RNA and ribosomal RNA were synthesized in both cultures during the 20 min pulse. Furthermore, the rate of ribosomal RNA synthesis and processing into functional ribosomes approached the rate reported for vegetative cells. Initial sporulation conditions which caused a prolonged delay in the rise in medium pH adversely affected the kinetics of appearance and number of ascospores. The affect was shown to be on meiotic events since a reduction of sporulation was always accompanied by a reduction in the amount of intragenic recombination.

Ribonucleic acid synthesized in meiotic cells of Saccharomyces cerevisiae: effect of culture medium pH.

Pulse-labeled ribonucleic acid (RNA) was extracted from polysomes of sporulating cells of Saccharomyces cerevisiae and characterized in sucrose gradients and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transfer RNA, ribosomal RNA, and heterodisperse RNA, presumed to be messenger RNA, were synthesized during a 20-min pulse at T4 and T6 when labeling was performed in sporulation medium adjusted to pH 6.0. Furthermore, ribosomal RNA was processed into functional ribosomes during the pulse. The specific activity of pulse-labeled RNA of cells labeled in sporulation medium where the pH was unadjusted at T4 (pH 7.8) and T9 (pH 8.6) was 20- to 50-fold lower than RNA from cells labeled at pH 6.0. The low specific activity resulted from a 50-fold reduction in uptake of labeled precursors when the medium pH was greater than 7.2. However, heterodisperse RNA ranging from 4-17S in size and transfer RNA were synthesized during the pulse at T4 (pH 7.8),but the low specific activity of ribosomal RNA prevented a thorough analysis of its synthesis. Cellular impermeability at T9 (pH 8.6) resulted in minimal uptake of label, and an analysis of pulse-labeled transcripts was impossible. A comparison of the percantage of polysomal material indicate, however, that these cells were at least as active in translation as cells pulse-labeled at pH 6.0.

Synthesis of polyadenylic acid-containing ribonucleic acid during the germination of Neurospora crassa conidia.

Ribonucleic acid (RNA) synthesized during the first 1 h of conidial germination (15 to 20, 25 to 30, and 55 to 60 min) has been characterized by sucrose-sodium dodecyl sulfate gradient centrifugation, binding to polyuridylic acid filters, and oligo(dT)-cellulose chromatography. At all labeling periods examined, polyadenylic acid-containing RNA is synthesized, processed, and incorporated into polysomes. Approximately 40% of the labeled RNA sedimenting between 5 and 17S binds to polyuridylic acid filters. RNA which binds to oligo(dT)-cellulose displays a heterogeneous distribution in sucrose-sodium dodecyl sulfate gradients with a major, broad peak at 10-16S. In addition, some polyadenylic acid-containing RNA sediments beyond the 25S marker. Approximately 3% of the [3H]adenosine in pulse-labeled polysomal RNA is in polyadenylic acid segments resistant to pancreatic and T1 ribonucleases.

Lomofungin, an inhibitor of ribonucleic acid synthesis in yeast protoplasts: its effect on enzyme formation.

Lomofungin, an antibiotic active against fungi, yeasts, and bacteria, rapidly inhibits synthesis of ribonucleic acid (RNA) but not protein by protoplasts of Saccharomyces strain 1016. With 40 mug of lomofungin/ml, RNA synthesis was almost completely halted after 10 min of incubation; protein synthesis continued for at least 40 min. Since lomofungin inhibits isolated RNA polymerases from yeast, but not in vitro protein synthesis, it is concluded that the primary action of lomofungin in yeast protoplasts is on RNA synthesis. Examination of the pulse-labeled RNA indicated that biosynthesis of both ribosomal precursor RNAs and messenger RNAs was severely inhibited after the protoplasts were incubated with lomofungin for 5 min, whereas formation of small-molecular-weight RNA (4 to 5s) was only slightly affected. Under these conditions, lomofungin almost completely prevented induction of alpha-glucosidase. Once the protoplasts had been induced, further production of the enzyme was not impaired by lomofungin until after 30 min of incubation, but was rapidly halted by cycloheximide (4 mug/ml). Lomofungin inhibition of invertase formation by protoplasts actively synthesizing the enzyme also became evident only after a lag of about 30 to 40 min, although synthesis was promptly halted by cycloheximide. These observations suggest the existence of relatively long-lived specific messenger RNAs for these enzymes.

Protein synthesis and deoxyribonucleic acid-membrane attachment during thymineless death in Escherichia coli.

The proteins synthesized during thymineless death in Escherichia coli B and B/r were analyzed by polyacrylamide gel elctrophoresis. It was found that the amount of a protein of molecular weight 80,000 to 88,000 is greatly increased during thymineless death compared to the amounts of other cell proteins. A technique for the isolation of cell membrane-deoxyribonucleic acid (DNA)-nascent ribonucleic acid (RNA) complex on detergent crystals was used to determine whether DNA might be detached from the cell membrane as a result of thymineless death. It was found that under no conditions of thymineless death or immunity to thymineless death was there any change in the attachment of DNA or pulse-labeled RNA to cell membrane.

Membrane-associated replication complex in arbovirus infection.

Cytoplasmic extracts of chicken embryo fibroblast cells infected with Semliki Forest virus were subjected to isopycnic centrifugation in discontinuous sucrose gradients. Seven distinct bands were usually formed. The four upper bands contained predominantly smooth membranes and the lowest band was enriched in rough endoplasmic reticulum. One fraction (fraction 5), banding at a density of 1.16 g/cm(3), was found to be heavily enriched in pulse-labeled ribonucleic acid (RNA), viral RNA polymerase, and viral RNA forms associated with RNA replication. Thus, fraction 5 evidently contained a membrane-associated viral replication complex of a type previously defined in picornavirus infections. Fraction 5 was also consistently enriched with unique membranous structures previously observed in intact cells as type 1 cytopathic vacuoles (CPV-1). When the CPV-1 in fraction 5 were isolated from cells briefly incubated with (3)H-uridine and (3)H-adenosine prior to cell disruption, a large proportion was found to be labeled by high-resolution autoradiography. Thus, ultrastructural, biochemical, and biological evidence were all consistent with the interpretation that the CPV-1 membranes represent a significant element of the viral replication complex.

Ribosomal ribonucleic acid synthesis and maturation in the blue-green alga Anacystis nidulans.

Methods are described for preparation of pulse-labeled ribonucleic acid (RNA) from the blue-green alga Anacystis nidulans. Synthesis of labeled RNA was found to be in part dependent on concurrent photosynthesis and was inhibited by the antibiotic streptolydigin. Mature 23S ribosomal RNA (rRNA) appeared before mature 16S rRNA. Formation of either molecule was inhibited by chloramphenicol, and RNA species of lesser mobility accumulated. These species may be precursors of the mature forms. Maturation of 16S rRNA was also inhibited by streptolydigin. (The effect of this antibiotic on 23S rRNA maturation was not examined). In many respects, ribosomal RNA synthesis and maturation in this blue-green alga appear to follow the pattern already established for bacteria.

Transcription after bacteriophage SPP1 infection in Bacillus subtilis.

The role of the host polymerase in Bacillus subtilis infected with phage SPP1 was studied in vivo with regard to production of phage-specific and host-specific ribonucleic acid (RNA) and to phage yield. Evidence is presented that the subunit(s) of B. subtilis RNA polymerase which is sensitive to rifampin and streptolydigin is necessary at all times during infection for phage production. The synthesis of phage RNA and the phage yield in strains resistant to either antibiotic were unaffected by the drug. Host RNA synthesis continued throughout infection; phage-specific RNA never accounted for more than 20% of pulse-labeled RNA at any time during infection.

Changes in ribonucleic acid turnover during aerobic and anaerobic growth in Rhodopseudomonas spheroides.

Differences in the rate and extent of degradation of ribonucleic acid (RNA) labeled by a 30-sec pulse in aerobically or anaerobically grown Rhodopseudomonas spheroides have been studied by using rifampin to block RNA synthesis. In anaerobic cultures, unstable RNA is degraded with a half-life of 1.25 to 2.0 min, and about 40% of the pulse-labeled RNA is stable. In aerobic cultures, the half-life of unstable RNA is increased to 2.5 to 4.0 min, and 50% of the RNA is stable. When aerobic cultures are transferred to anaerobic conditions, there is a rapid drop in half-life and in the proportion of stable RNA. When anaerobic cultures are made aerobic, the reverse changes occur after a lag of about 30 min. Addition of puromycin to either aerobic or anaerobic cultures caused the pulse-labeled RNA to be degraded at the same rate and to the same extent as the RNA in an anaerobic control culture. In contrast, addition of chloramphenicol enhanced the difference in RNA half-life and increased the proportion of stable RNA by about 10% in each case. It is concluded that there is a difference in the stability of an RNA component under aerobic and anaerobic conditions.

Ribonucleic acid from aerobically and anaerobically grown Rhodopseudomonas spheroides: comparison by hybridization to chromosomal and satellite deoxyribonucleic acid.

Ribonucleic acid (RNA) species from aerobically and anaerobically grown Rhodopseudomonas spheroides were compared via hybridization to deoxyribonucleic acid (DNA). Both long-labeled and stable RNA bound to chromosomal DNA to the same extent, regardless of derivation. About 4% of the chromosomal DNA hybridized with total cell RNA and about 0.08% with stable RNA. About 4% of the mixed satellite DNA could be hybridized to total cell RNA from aerobic or anaerobic cultures, whereas essentially no stable RNA formed a hybrid with this DNA. Hybridization competition experiments with aerobic and anaerobic pulse-labeled RNA and chromosomal or satellite DNA demonstrated that no qualitative differences existed between the RNA species. It is concluded that identical species of RNA in the same relative amounts are synthesized by R. spheroides during aerobic or anaerobic growth on the same medium.

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.

Alterations in the distribution of labeled ribonucleic acid in polyribosomes from Escherichia coli infected with bacteriophage R17.

The distribution of labeled ribonucleic acid (RNA) associated with polysomes from Escherichia coli infected with the bacteriophage R17 was investigated. Pulse-labeling of RNA for 15 sec with (3)H-uridine resulted in increased labeling of the RNA associated with larger polysomes from infected cells as compared to control cells. Analysis of the RNA indicated that the increased labeling of large polysomes resulted from the presence of labeled double-stranded viral RNA. Other species of 15-sec pulse-labeled RNA entered into polysome formation in both infected and control cells. On the other hand, pulse-labeling of cultures for 15 sec with (3)H-uridine followed by a 5-min chase with unlabeled uridine resulted in a greater decrease in the amount of labeled RNA associated with large polysomes from infected cells as compared to control cells. This decreased labeling of large polysomes from infected cells was accompanied by an increased amount of label associated with the monomer to trimer regions. Analysis of RNA labeled under pulse-chase conditions indicated that virus infection resulted in an increased amount of heterogeneous 5 to 15S RNA in both the monomer to trimer and ribosomal subunit-soluble regions of the polysome profile. Labeled 5 to 15S RNA extracted directly from infected cells under pulse-chase conditions, without prior polysome fractionation, was characterized by a shift toward a distribution of smaller polynucleotides.

Asymmetric Template Function of Microbial Deoxyribonucleic Acids: Transcription of Messenger Ribonucleic Acid

In Bacillus subtilis and Escherichia coli, pulse-labeled ribonucleic acid (RNA) synthesized during step-down growth hybridized preferentially with the heavy (H) strand of methylated albumin-Kieselguhr-fractionated deoxyribonucleic acid (DNA). At high RNA inputs, the ratio of RNA hybridized with the H strand to that hybridized with the light (L) strand was 8.7 for B. subtilis and 2.0 for E. coli. At high DNA inputs, the H/L hybridization ratio increased by a factor of two. This change in the hybridization ratio was attributable to the fraction of the pulse-labeled RNA which is in stable RNA components. The hybridization peak of pulse-labeled RNA was specifically located in the late-eluting region of the absorbance profile of the H strand. This region was considered to represent the most actively transcribing H strand templates.

Effect of Elevated Temperature on Extended Enzyme Synthesis Induced by Bacteriophage T4 Amber Mutants Unable to Synthesize Deoxyribonucleic Acid

The extended synthesis of early enzymes by the deoxyribonucleic acid-negative amber mutants of bacteriophage T4 after infection of the nonpermissive host Escherichia coli B was prevented by incubating the infected cells at 44 C. This effect did not occur if the incubation temperature was 43 C or less or if the cells were grown and infected in broth rather than minimal medium (C medium). Once early enzyme synthesis had ceased at 44 C, lowering the incubation temperature to 37 C did not occasion resumption of synthesis. Experiments with chloramphenicol at 44 C indicated that increased degradation of early enzymes is an unlikely explanation for the effect. Examination of pulse-labeled ribonucleic acid and polysomes made at 37 and 44 C in infected cells revealed some differences, but at present there is no obvious way in which these differences may be related to the effect on enzyme formation. There was no discernible difference between the ribosomal ribonucleic acid and ribosomes at the two temperatures, nor was there a difference in the cell-free amino acid-incorporating systems isolated from cells infected at the two temperatures as judged by polyuridylic stimulation of phenylalanine incorporation. Incubation of cells infected with T4amN82 at 44 C with protein synthesis blocked by 5-methyltryptophan for 15 min did not prevent the typical pattern of enzyme synthesis at 44 C when the block was reversed by excess l-tryptophan. The relation of this and other observations relative to the effect at 44 C on the synthesis of early enzymes is discussed.

Pulse-labeled ribonucleic acid complexes released by dissociation of rat liver polysomes.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPulse-labeled ribonucleic acid complexes released by dissociation of rat liver polysomesSe Yong Lee and George BrawermanCite this: Biochemistry 1971, 10, 3, 510–516Publication Date (Print):February 1, 1971Publication History Published online1 May 2002Published inissue 1 February 1971https://doi.org/10.1021/bi00779a025RIGHTS & PERMISSIONSArticle Views22Altmetric-Citations28LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (658 KB) Get e-Alerts Get e-Alerts

Complementary Strands of Bacteriophage φ29 Deoxyribonucleic Acid: Preparative Separation and Transcription Studies

Bacillus subtilis phage phi29 has a nonpermuted, duplex deoxyribonucleic acid (DNA) with cohesive ends and a molecular weight of 11 x 10(6). Denaturation of this DNA yielded two intact polynucleotide chains. Preferential binding of the polyribonucleotide polyuridylic-guanylic acid (poly UG) to the complementary strands of denatured phi29 DNA permitted separation of the strands in neutral CsCl gradients. In analytical CsCl density gradient centrifugation, the separated strands with poly UG appeared as two symmetrical bands, both heavier than the normal denatured DNA band. The strands differed in density by 11 mg/cc. Preparative separation of the phi29 DNA strands resulted in two fractions, heavy (H) and light (L). The H fraction was essentially free from L contamination, whereas L contained up to 25% of H, as determined both by rebanding the separated fractions in CsCl and by electron microscopic examination of self- and mixed-annealed fractions. Pulse-labeled ribonucleic acid (RNA) prepared at intervals after infection was hybridized with the self-annealed DNA strands. Preliminary experiments indicated that both strands of phi29 DNA are transcribed during the development of the virus. Early transcribed phi29-specific RNA hybridizes only with the L strand; at later times, transcription occurs from both the L and H strands.

Transient stimulation of deoxyribonucleic acid-dependent ribonucleic acid polymerase and histone acetylation in human embryonic kidney cultures infected with adenovirus 2 or 12: apparent induction of host ribonucleic acid synthesis.

The synthesis of cell-specific ribonucleic acid (RNA) appeared to be stimulated in human embryonic kidney (HEK) cultures infected with adenovirus 2 or 12. Deoxyribonucleic acid (DNA)-RNA hybridization experiments revealed that by 44 to 70 hr after infection with either virus, the relative amount of pulse-labeled RNA capable of hybridizing with HEK cell DNA increased considerably; such RNA was detected in both nuclear and cytoplasmic fractions. The main increase in apparent host RNA synthesis was preceded by (i) a relatively early transient stimulation of the DNA-dependent RNA polymerase activity in isolated nuclei, and (ii) a small but consistently observed increase in the rate of acetylation of lysine-rich and arginine-rich histone fractions. The Mn(2+)-(NH(4))(2)SO(4) and Mg(2+)-activated RNA polymerase reactions measured in nuclei isolated from cells infected with adenovirus 2 or 12 were stimulated at about the same time; a rapid loss of polymerase activity followed. The augmentation of the two RNA polymerase reactions found in adenovirus 12-infected cells was independent of protein synthesis. After the initial increase, the acetylation rate of histones of cells infected with adenovirus 2 or 12 declined, until late in infection it was approximately 40 to 70% of the control cell rate.

Stable messenger ribonucleic acid and germination of Myxococcus xanthus microcysts.

We have examined germination, protein synthesis and ribonucleic acid (RNA) synthesis by microcysts of the fruiting myxobacterium Myxococcus xanthus. The morphological aspects of microcyst formation were completed at about 2 hr after induction had begun. In such microcysts, germination, RNA synthesis, and protein synthesis were inhibited by actinomycin D (Act D). At 6 hr after induction, germination and protein synthesis had become relatively resistant to Act D, whereas RNA synthesis was inhibited by about 95%. Experiments with (3)H-Act D indicated that the deoxyribonucleic acids of both young and old microcysts bind Act D equally. Resistance of germination to Act D was acquired 4 to 5 hr after induction of microcyst formation, and was due to an Act D-sensitive synthesis at that time. Vegetative cells and microcysts were pulsed with uridine-5-(3)H and chased for 60 min; the RNA was extracted and analyzed by means of sucrose density gradient centrifugation and gel electrophoresis. Both microcysts and vegetative cells were found to contain grossly the same types of RNA in the same proportions. RNA pulse-labeled in microcysts was more stable than that in vegetative cells. No particular portions of the microcyst pulse-labeled RNA were selectively stabilized. These data indicate that a stable messenger RNA required for synthesis of germination proteins was synthesized during microcyst formation. This may be the same as the RNA synthesized 4 to 5 hr after initiation of microcyst formation. We suggest that the existence of such stable messenger RNA in microcysts is consistent with the limited biosynthetic activities of such cells.

Control of Gene Function in Bacteriophage T4

Deoxyribonucleic acid (DNA) synthesis in T4rII-infected, lambda-lysogenic strains of Escherichia coli proceeds with one-half the rate of T4 wild-infected bacteria and stops 16 min after infection at 37 C. The rates of ribonucleic acid (RNA) synthesis, however, are the same with T4rII and T4 wild. The turnover of pulse-labeled RNA is slow in K strains (half-lives 10 to 20 min) as compared with B strains (half-lives 2.5 to 6 min). Lambda-lysogeny increases the apparent messenger (m) RNA half-lives in pulse-chase experiments. The shutoff of host RNA synthesis in T4rII infected K(lambda) is incomplete. Moreover, the preferential transcription of T4 DNA ceases 13 min after infection, and transcription of host and prophage lambda DNA is resumed. The T4 RNA synthesized in rII-infected K(lambda) contains no late T4 mRNA. The early portion of the T4 genome, however, is transcribed completely. The T4-induced early modification of bacterial RNA polymerase does occur. Resumption of host DNA transcription at 13 min after infection is not associated with a reversal of the above polymerase modification. It is concluded that in lambdalysogenic bacteria T4rII infections are abortive because RNA polymerase is prevented from transcribing late T4 genes.