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An Escherichia coli translation system supplemented with ribonucleic acid from sporulating Bacillus subtilis produces unique polypeptides which are missing among translation products of ribonucleic acid from six early sporulation mutants.
A procedure is described for isolation in a soluble form of a ribonucleic acid polymerase from rat testes. Evidence is presented that this enzyme catalyzes three distinct reactions: (a) deoxyribonucleic acid-directed synthesis of RNA in the presence of all four major ribonucleoside triphosphates; (b) DNA-primed formation of polyadenylic acid and other ribohomopolymers in the presence of single ribonucleoside triphosphate substrates; (c) synthesis of complementary polyribonucleotides in the presence of various ribohomopolymer primers. The properties of these reactions are discussed with respect to metal ion requirements, affinities for ribonucleoside triphosphates and primer polynucleotides, heat denaturation of DNA primers, and the effects of ionic strength, beta-mercaptoethanol, polyamines, temperature, and inhibitors on the rates and extent of the reactions. The testicular ribonucleic acid polymerase is a very unstable enzyme that can be stabilized by high concentrations of glycerol.
Morphological studies of a conditionally temperature-sensitive ribonucleic acid polymerase mutant of Bacillus subtilis have revealed that sporulation is inhibited at stage II when the cells are grown at 47.5 C. Growth and sporulation occur normally at 30 C with the mutant. The mutant grows normally at 47.5 C but is prevented from sporulating at the nonpermissive temperature by an abnormal septation during forespore membrane formation which prevents the subsequent engulfment process (stage III). The mutation affects the normal functioning of ribonucleic acid polymerase at the nonpermissive temperature resulting in abortive sporulation.
The levels of protein and ribonucleic acid in the cerebrum, cerebellum, optic lobes and medulla oblongata of normal and alloxan-diabetic rats were measured. In general, the protein content and levels of ribonucleic acid in the broad compartments of the brain of rat decreased during diabetes.
OBJECTIVE: The goal of this study was to develop and validate a cytochrome P450 (CYP) 2D6 probe substrate with improved sensitivity to elucidate the relationship of CYP2D6 ribonucleic acid transcript levels, genotype, and enzyme activity in human liver biopsy samples. METHODS: CYP2D6 activity in tissue homogenates of liver biopsy specimens collected from control subjects (with no apparent liver disease), liver biopsy subjects, liver transplant subjects, and liver bank specimens was assessed with a calcimimetic, R-568, a high-clearance and specific substrate of CYP2D6. The livers were genotyped for the 6 most common CYP2D6 genetic variants (ie, *3, *4, *5, *6, *7, and *8). The 1.5-kilobase CYP2D6 messenger ribonucleic acid (referred to as full-length) transcripts were estimated with a semiquantitative reverse transcription-polymerase chain reaction assay. RESULTS: As a CYP2D6-specific catalytic probe, R-568 offers a 20-fold higher sensitivity compared with that of dextromethorphan. The improved assay sensitivity allowed evaluation of CYP2D6 enzyme activity in a few milligrams of tissue collected from biopsy specimens. The ratio of CYP2D6 enzyme activity to transcript remained relatively constant within each group of subjects, especially within the control group. However, mean activity to transcript varied greatly across the 4 groups of subjects. The liver samples in the control group showed significantly higher enzyme activity but a lower transcript level. CONCLUSIONS: A combination of genotyping and messenger ribonucleic acid level determination could allow a quantitative estimation of functional CYP2D6 activity in healthy human livers with a reasonable degree of confidence. Kinetic study with R-568 indicates that this compound is probably the most sensitive CYP2D6 probe substrate available.
Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (r-protein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 42 degrees C; in recA strains, the degradation took place at 42 degrees C but was very slow or absent at 30 degrees C. The recA strains carrying pNO1001 failed to form colonies at 30 degrees C, presumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process.
Henney, H. (The University of Texas, Austin) and R. Storck. Nucleotide composition of ribonucleic acid from Neurospora crassa. J. Bacteriol. 85:822-826. 1963.-The nucleotide composition of total, ribosomal, and soluble ribonucleic acid (RNA) from the conidia and the hyphae of Neurospora crassa was determined. The corresponding RNA classes from the two morphological types had the same base ratio. Total, ribosomal, and soluble RNA from hyphae contained, respectively, 51.0, 50.2, and 61.6% guanylic acid (G) plus cytidylic acid (C), and from conidia, 50.6, 49.9, and 62.1%. The proportion of nucleotides in ribosomal RNA was in close agreement with that reported for ribosomal RNA from other organisms. Soluble RNA contained 2.8% pseudouridylic acid, 36.0% C, and 25.5% G, and differed from soluble RNA from other sources in its high C content. Centrifugation of purified RNA from conidia and hyphae, through a linear sucrose-density gradient, yielded identical sedimentation profiles.
OBJECTIVE: Our purpose was to identify molecular mechanisms underlying abnormal growth of uterine leiomyomas. STUDY DESIGN: Biopsy samples of tumor and adjacent "normal" myometrium from nine patients were analyzed for progesterone receptor gene expression and for proliferation-associated antigen Ki-67. RESULTS: Northern analysis indicated that progesterone receptor messenger ribonucleic acid levels were increased twofold to 15-fold in leiomyoma compared with adjacent myometrial biopsy tissue from all patients (n = 9), whereas beta-actin messenger ribonucleic acid was at similar levels in these samples. Quantitative immunoassay, immunohistochemistry studies, and Western blot analyses revealed increased amounts of progesterone receptor protein in the tumor tissue. Both the progesterone receptor A and B forms were expressed in the leiomyoma and adjacent myometrium. Corresponding to increased progesterone receptor gene expression, the proliferation-associated antigen Ki-67 was also significantly elevated in the leiomyoma tissue. CONCLUSION: These data provide the first evidence that progesterone receptor messenger ribonucleic acid is overexpressed in uterine leiomyomas, suggesting that amplified progesterone-mediated signaling is instrumental in the abnormal growth of these tumors.
OBJECTIVES: This study was undertaken to determine expression levels of oxytocin receptor and its gene in peri-implantation phase human endometrium during clomiphene-treated cycles compared with control cycles. STUDY DESIGN: Oxytocin receptor and its messenger ribonucleic acid in peri-implantation phase endometrium during control and clomiphene-treated (50 mg days 5 to 9) cycles of 5 healthy fertile women were determined by immunohistochemical methods, Western blot analysis with monoclonal antibody against amino acids 20 through 40 of the extracellular N-terminal human oxytocin receptor, and reverse transcription-polymerase chain reaction with oligonucleotide primers to amplify the 391-base pair fragment of the oxytocin receptor gene. RESULTS: Oxytocin receptor and its messenger ribonucleic acid were expressed in human peri-implantation phase endometrial samples from both control and clomiphene-treated cycles. The receptor was localized predominantly in the epithelial cells and glands, with little or none detected in the stroma. Oxytocin receptor protein was separated out as a single 70-kd band by Western blot analysis; its relative abundance was significantly reduced during clomiphene-treated cycles. The messenger ribonucleic acid was detected in all endometrium during control and clomiphene-treated cycles, with greater expression during control cycles. CONCLUSIONS: The expressions of oxytocin receptor and its gene in luteal phase human endometrium suggest a functional relevance in modulation of biochemical changes for implantation.
Previously, virtually complete 16S ribosomal ribonucleic acid sequences were determined for 54 strains of species in the family Pasteurellaceae. The sequences for 16 additional strains have been determined, bringing the total number of strains sequenced to 70. The additional strains include: Actinobacillus hominis, A. muris, A. salpingitis, Pasteurella bettyae, P. mairii, P. testudinis, and Bisgaard taxa 2, 3, 5, 6, 7, 8, 9, 13, and 14 (2 strains). A phylogenetic tree was constructed based upon sequence similarity using the Neighbor-Joining method. The additional sequence information and phylogenetic analysis generally supported our previously described phylogenetic structure for the family Pasteurellaceae. Cluster 1, containing Haemophilus sensu stricto, was unchanged. P. mairii was closely related to P. aerogenes and Bisgaard taxon 6 was related to H. somnus in Cluster 2. A. salpingitidis and Bisgaard taxa 2, 3, 7, and 13 fell in Cluster 3 which contains Pasteurella sensu stricto. A. hominis was closely related to Actinobacillus sensu stricto species in Cluster 4A. Bisgaard taxa 5, 8, 9 and P. bettyae fell in Cluster 4B. A. muris was related to P. pneumotropica in Cluster 5. Haemophilus parainfluenzae strains branched deeply as a 6th cluster. Bisgaard taxon 14 and P. testudinis formed a 7th cluster which branched deeper than any previously described clusters in the family Pasteurellaceae. The branching was extremely complex and taxonomic division of the family into phylogenetically and phenotypically coherent genera will be difficult.
Rat liver nucleocytosolic messenger ribonucleic acid (mRNA) transport is shown to be regulated by proteins with a high affinity for nucleic acids. In the cell-free system described, the energy-dependent transport of all RNA classes [transfer RNA (tRNA), mRNA, and ribosomal RNA (rRNA)] exhibited a dependence upon the availability of discrete minor sets of cytosol proteins. In addition to having a different level of saturation, only the mRNA "transport protein" activities are increased by adenosine cyclic 3',5'-phosphate (cAMP), an effect most likely mediated by a cAMP-dependent protein kinase. The mRNA transport proteins were isolated from cytosol by precipitation with streptomycin sulfate followed by deoxyribonucleic acid (DNA)-cellulose affinity chromatography, or from oligo-(thymidylate)-cellulose bound cytoplasmic messenger ribonucleoprotein (mRNP) particles by high-salt extraction. Either method yielded a protein fraction which exhibited a 1000-fold increase in mRNA transport activity as compared to cytosol. Over one-half of the mRNA transport activity is associated with the mRNP of the cell. A partial homology between the cytosol and mRNP-derived proteins was demonstrated by polyacrylamide gel electrophoresis. One major (20 000 daltons) and several minor proteins (23 000, 52 000, 54 000, and 72 000 daltons) were in common. Nuclear 4-5S exited from in vitro incubated nuclei in three phases, according to their differential in vivo rates of labeling and intranuclear pool sizes. The amount of nuclear RNA transported in vitro as mRNA (about 1.0%) agrees wtih the in vivo estimates. Additional evidence for in vivo equivalence was provided by the physicochemical characterization and bioassay of the RNA. The transported mRNA sedimented in urea-sucrose gradients as an 8-18S heterodisperse product. This RNA initiated cell-free translation with the synthesis of precursor peptides as diverse in size as those for albumin and alpha 2U-globulin. The relative abundancies of various transported mRNAs were different than the corresponding abundancies of liver cytoplasmic mRNAs.
Messenger ribonucleic acid (mRNA) for thymus-leukemia antigens, membrane-associated glycoproteins of murine leukemia cells, was obtained from polysomes of murine leukemia cells forming thymus-leukemia antigens. Polysomes forming thymus-leukemia antigens were recovered by immunoprecipitation using alloantibodies specific for the 1,2,3 determinants of the thymus-leukemia antigen complex before they were extracted with phenol-detergent. Poly(adenylic acid)-containing RNA [poly(A)-RNA] was fractionated by oligo[deoxythymidylate] [oligo(dT)]-cellulose chromatography. The mRNA obtained had a sedimentation coefficient of approximately 17 S in agreement with the predicted size necessary for forming proteins specifying thymus-leukemia antigens. In a wheat germ system, the polypeptides formed upon addition of mRNA for thymus-leukemia antigens consisted of a major product with a molecular weight of 42,000. It was larger than the nonglycosylated heavy chain molecule of 40,000 daltons formed by the cells themselves. On the cell surface thymus-leukemia antigens exist as glycosylated molecules of 47,000 daltons associated with a light-chain equivalent to beta 2-microglobulin. Molecules of 40,000 daltons were isolated from the cells cultured in the presence of tunicamycin, an inhibitor of de novo glycosylation, and by treating thymus-leukemia heavy chains with endo-beta-N-acetylglucosaminidase H.
The region of the ovalbumin messenger ribonucleic acid (mRNAov) molecule bound to the 40S ribosomal subunit and its associated initiation factors in the wheat germ cell-free translation system were isolated and characterized. Two mRNAov fragments, 87 and 92 nucleotides in length, were protected from T1 ribonuclease digestion by binding of guanosine 5',beta,gamma-methylenetriphosphate and were shown by hybridization and fingerprint mapping to be derived from the 5' end of mRNAov. Both these mRNAov fragments were of sufficient length to contain both the cap structure and the AUG initiation codon. Four T1-resistant oligonucleotides, prepared by direct digestion of mRNAov with T1 ribonuclease were also found to bind to the wheat germ 40S ribosomal subunit. Nucleotide sequence analysis of these oligonucleotides revealed (1) that they were not a subset of the ribosome binding fragments described above, (2) that they were derived from within the mRNAov molecule (one from within the coding region and three from the noncoding region at the 3' end of the mRNAov molecule), and (3) that three of the four mRNAov nucleotides contained 3'-terminal AUG trinucleotides. These data suggested that features of the mRNAov molecule in addition to the nucleotide sequence might be important in specifying the correct ribosome binding site for the initiation of protein synthesis. The amount of mRNAov bound to the wheat germ 40S ribosomal subunit in a preinitiation complex was found to vary inversely with the potassium ion concentration. Lowering the potassium concentration to levels suboptimal for translation also resulted in the protection of larger fragments of the mRNAov molecule derived from the same 5'-end region as the ribosome binding fragments described above. The ability of the cap analogue 7-methylguanosine 5'-phosphate (m7G5'p) to reduce the amount of mRNAov bound to the wheat germ 40S ribosomal subunit was found to depend directly on thepotassium concentration. Interestingly, the effects of potassium on the amount of mRNAov bound in a preinitiation complex and the inhibition of this binding by m7G5'p could be observed by changing the potassium concentration after binding had occurred. These data suggested that the interaction between the wheat germ 40S ribosomal subunit and mRNAov was very sensitive to the ionic environment.
Transfer ribonucleic acid(1) is methylated after the molecule is synthesized; at least eight enzymes are involved in the transfer of methyl groups (derived from methionine). The time courses of methylation and synthesis of tRNA during rat liver regeneration have been compared in an in vivo radioisotopic study, using 6-orotic acid-(14)C and (3)H-methyl-L-methionine as precursors in double label pulses. Liver regeneration is a synchronized system in which biochemical events of the cell cycle are separable. Transfer RNA methylation increase precedes by several hours tRNA synthesis during regeneration, although the curves overlap. A ratio of the relative rate of methylation to the relative rate of synthesis has been made; that curve positively correlates with the rise and fall of protein synthesis during regeneration. It is clear that methylation and synthesis of tRNA are only weakly coupled; changing methyl content of the tRNA "pool" resulting from differential tRNA methylase and polymerase activities may regulate the rate of protein synthesis in the cell cycle at the translational level. The "pool sizes" of uridine monophosphate (UMP) and S-adenosylmethionine (SAM) were measured indirectly; UMP and SAM were isolated from perchloric acid supernatants and their specific activities were computed. Differential changes in radioactivity available to tRNA methylases and polymerases are not a source of artifact. That is, the control of both the synthesis and methylation of tRNA is at the enzyme level in vivo, rather than at some enzymatic step prior to those enzymatic reactions.
Ribonucleic acid prepared by the method of Gierer and Schramm from concentrated and partially purified types I and II polioviruses has been demonstrated to be infectious for HeLa and human amnion cells in monolayers. In areas of cytopathogenic action resulting from invasion of cells by RNA, intact poliovirus, of the type from which the RNA had been prepared, is present. The infectivity of the RNA was completely inactivated by a 2 minute exposure to purified ribonuclease or to whole normal monkey serum shown to contain measurable concentrations of this enzyme. Whole virus infectivity was not influenced by RNAase or whole normal monkey serum. Normal and polio-immune globulin, desoxyribonuclease, lysozyme, proteolytic enzymes, and bovine albumin failed to inactivate the infectivity of RNA. The degree of infectivity of isolated RNA from poliovirus for cells in monolayer was greatly influenced by the ionic strength of the environment. The experimental evidence suggests that isolated poliovirus RNA is the carrier of the biological activity responsible for infection of cells and for transmission of genetic information which controls type specificity.
Cytoplasmic initiator transfer ribonucleic acid (tRNAinit) was purified from bulk Torulopsis (Candida) utilis tRNA by a series of column chromatography procedures. Sequence analysis of the products of complete and partial digestion of this tRNA with ribonuclease A [EC 3.1.4.22] and ribonuclease T1 [EC 3.1.4.8] enabled us to determine the complete primary structure of the molecule. The chain length of this tRNA was 76, including 11 modified nucleotides. The structure of the tRNA was arranged into a cloverleaf model and compared with those of other initiator tRNA species. As in the cytoplasmic initiator tRNA's of most other eukaryotic cells, the sequence -A-U-C-G- is contained in this tRNA in place of the usual -T-psi-C-G (or A)- found in other tRNA's.
Messenger ribonucleic acid (mRNA) for actin was detected in celloidin-embedded archival human temporal bone sections with reverse transcription polymerase chain reaction (RT-PCR). Actin mRNA was detected in 10% of sections analyzed. One possible reason for this modest detection incidence is enzymatic degradation of RNA by exogenously introduced ribonucleases (RNases). We have identified steps of the temporal bone processing protocol for archival storage in which exogenous RNases could be introduced to the tissue, and have verified that the bone sections are exposed to these enzymes. We have demonstrated that implementing precautions to minimize exogenous RNase contamination during processing improves recovery of intact RNA. This study indicates that although gene expression analysis of archival human temporal bones may be limited by enzymatic degradation of RNA, simple modification of processing protocol can improve yield of informative data.
Double-stranded ribonucleic acid (ds-RNA) isolated from Escherichia coli infected with bacteriophage MS2 is a potent interferon inducer. High levels of ds-RNA are formed in nonpermissive cells infected with MU9, an amber coat protein mutant of MS2. This mutant has been used to develop a process for large-scale ds-RNA production. Preparation of quantities of MU9 lysate sufficient for ds-RNA production in fermentors is described. Over 300 mug of ds-RNA/ml can be accumulated after MU9 infection of cultures grown to high density in corn steep liquor medium. This is approximately 300 times the amount of ds-RNA made by MS2 infection of cells grown in tryptone medium. Maximum ds-RNA formation requires only 3 hr. The ds-RNA is stable and remains inside nonaerated cells for at least 17 hr.