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The plaque titer of poliovirus "ribonucleic acid" on monkey kidney cells cultured in vitro is greatly increased by depleting these cells of calcium and treating the "ribonucleic acid" inoculum with a suspension of any one of several poorly water-soluble substances before inoculation. These undissolved substances apparently facilitate infection by serving as solid vehicles for the "ribonucleic acid."
In Bacillus subtilis and Escherichia coli, 16 and 23S ribosomal ribonucleic acid (rRNA) hybridize exclusively with the heavy (H) strand of methylated albuminkieselguhr (MAK)-fractionated complementary deoxyribonucleic acid (DNA) strands. All the soluble RNA (4S RNA) in B. subtilis and 66 to 75% of the 4S RNA in E. coli also hybridize with the H strand. Interspecific hybridization shows that E. coli 23S rRNA also binds selectively to the DNA H strand of Salmonella typhimurium. The hybridization peak for all three cellular RNA components is specifically located in the late-eluting region of the absorbance profile of the DNA H strand. The early-eluting region of the light (L) strand preferentially inhibits the hybridization between the peak region of the H strand and 23S rRNA. These regions are considered to represent the transcribing sequences and their complements for 23S rRNA in the separated H and L strands of DNA, respectively.
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It appears that a de novo synthesis of the deoxyribonucleic acid-dependent ribonucleic acid-polymerase in Bacillus cereus T takes place fairly late in outgrowth, at the onset of the vegetative cycle. Therefore, the ribonucleic acid-polymerase used by germinating spores is the one carried on from sporulating cells. However, the sporal enzyme is less soluble that the vegetative one, and its "core" is bound to two extra peptides. This complexing to other molecules could play a role in the regulation of gene expression during germination.
A new approach for measurement of ribonucleic acid is based on extraction of ribonucleic acid by combined action of a detergent and high speed homogenization followed by precipitation of ribonucleic acid with cadmium +2 ions and its subsequent measure with orcinol. Disappearance of digesta ribonucleic acid flowing along the small intestine was measured in six rams, each equipped with three T-shaped cannulas that were placed in six sites of the small intestine. Chromic oxide was a marker to measure rate of flow of digesta. Net disappearance of ribonucleic acid was completed 7 m from the pylorus, amounting to 83.9% of the quantity which entered the duodenum. About 50% of the net degraded ribonucleic acid disappeared in the 1st m of the small intestine.
A cell cycle mutant strain which is defective in the G1 period, B2-39, was selected from 1,200 temperature-sensitive mutants of the heterobasidiomycetous yeast Rhodosporidium toruloides M-1057. In the mutant cells, ribosomal ribonucleic acid synthesis was initially inhibited upon temperature shift-up from a permissive (25 degrees C) to a restrictive (36 degrees C) temperature. Moreover, the mutant was found to be temperature sensitive in deoxyribonucleic acid-dependent ribonucleic acid polymerase I activity in vitro. In a revertant-mutant strain, B2-39-R-2, both ribosomal ribonucleic acid synthesis in vivo and enzyme activity in vitro were simultaneously recovered. These results indicate that the mutant has a temperature-sensitive, deoxyribonucleic acid-dependent ribonucleic acid polymerase I and suggest that ribosomal ribonucleic acid synthesis acts as one of the control factors for initiation of both deoxyribonucleic acid synthesis and bud emergence.
With a microcytotoxicity assay it was shown that normal, non-immune human lymphocytes were converted to effector cells specifically cytotoxic to human renal carcinoma cells after incubation with xenogeneic immune ribonucleic acids. The ribonucleic acid was extracted from the lymphoid tissues of sheep that had been immunized with human renal carcinoma tissue. Lymphocytes incubated without ribonucleic acid from or with ribonucleic acid sheep immunized with Freund's adjuvant alone did not increase cytotoxicity. Immunotherapy with immune ribonucleic acid increased cytotoxic activity of lymphocytes from a patient with metastatic renal carcinoma. The microcytotoxicity assay may be a useful method to assessing the cellular immune response in patients receiving immunotherapy and seems to correlate with their clinical course.
Regulation of isoleucine, valine, and leucine biosynthesis and isoleucyl-, valyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined in two mutant strains of Escherichia coli. One mutant was selected for growth resistance to the isoleucine analogue, ketomycin, and the other was selected for growth resistance to both trifluoroleucine and valine. Control of the synthesis of the branched-chain amino acids by repression was altered in both of these mutants. They also exhibited altered control of formation of isoleucyl-tRNA synthetase (EC 6.1.15, isoleucine:sRNA ligase, AMP), valyl-tRNA synthetase (EC 6.1.1.9, valine:sRNA ligase, AMP), and leucyl-tRNA synthetase (EC 6.1.1.4, leucine:sRNA ligase, AMP). These results suggest the existence of a common element for the control of these two classes of enzymes in Escherichia coli.
Six thionucleosides found in Bacillus subtilis transfer ribonucleic acids were investigated: N6-(delta 2-isopentenyl)-2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, 2-methylthioadenosine, N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine, and one unknown (X1). The presence of N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine was demonstrated based on the affinity of the transfer ribonucleic acid containing it for an immunoadsorbent made with the antibody directed toward N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine. The existance of N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine in two species of lysine transfer ribonucleic acids was also confirmed by high-resolution mass spectrometry. Four of these thionucleosides--N6-(delta 2-isopenenyl)-2-methylthioadenosine, 2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, and the unknown designated X1--occurred only in specific areas in the elution profile of an RPC-5 column and probably affect the chromatographic properties of the transfer ribonucleic acids containing them. In contrast with Escherichia coli, where 4-thiouridine is the most frequent type of sulfur-containing modification, approximately one-third of the sulfur groups in B. subtilis transfer ribonucleic acid are present as thiomethyl groups on the 2 position of an adenosine or modified adenosine residue.
The incorporation of [3H]thymidine into deoxyribonucleic acid by exponentially growing cells of Bacillus brevis was inhibited by streptolydigin and rifampin in the same concentration range in which these drugs inhibit ribonucleic acid synthesis. Complete inhibition occurred within one-third generation time after drug addition, suggesting an effect on deoxyribonucleic acid chain elongation.
The in vitro product of mouse leukemia virus deoxyribonucleic acid (DNA) polymerase can be separated into two fractions by sedimentation in sucrose gradients. These two fractions were analyzed for their content of single-stranded DNA, double-stranded DNA, and DNA-ribonucleic acid (RNA) hybrid by (i) digestion with enzymes of known specificity and (ii) equilibrium centrifugation in Cs(2)SO(4) gradients. The major fraction early in the reaction contained equal amounts of single-stranded DNA and DNA-RNA hybrid and little double-stranded DNA. The major fraction after extensive synthesis contained equal amounts of single-and double-stranded DNA and little hybrid. In the presence of actinomycin D, the predominant product was single-stranded DNA. To account for these various forms of DNA, we postulate the following model: the first DNA synthesis occurs in a replicative complex containing growing DNA molecules attached to an RNA molecule. Each DNA molecule is displaced as single-stranded DNA by the synthesis of the following DNA strand, and the single-stranded DNA is copied to form double-stranded DNA either before or after release of the single strand from the RNA. Actinomycin blocks this conversion of single-to double-stranded DNA.
In previous mutational studies with mutant trpA46 (Gly [GGA] --> Glu [GAA] at position 211 of the tryptophan synthetase alpha chain) of Escherichia coli, no missense suppressors were detected. Such suppressors have now been obtained by single mutations in gly Vins, the structural gene for a GGA/G-reading, mutationally altered form of gly V transfer ribonucleic acid (tRNA) (tRNA(Gly) which reads GGU/C). A trpA46 strain containing the gly Vins alteration was mutagenized with hydroxylamine, and suppressor mutations were detected in the prototrophs obtained. Eighteen independent suppressors were examined and shown to have alterations which map in the gly V region. Chromatography of the glycyl-tRNAs of one suppressed mutant on a benzoylated diethylaminoethyl-cellulose column revealed an alteration in the tRNA(ins) (Gly) peak. The trpA46 suppressor mutation thus appears to involve a change of tRNA(ins) (Gly) from a GGA/G (Gly) reader to a GAA (Glu) reader. Since this suppressor presumably retains the "wobble" pairing of gly Vins tRNA, it was used to select the conversion of GAU (Asp211) to GAG (Glu211) in the alpha chain. supD (serine-inserting amber suppressor) was then used to obtain the conversion of GAG (Glu211) to UAG211. Missense revertants of trpA (UAG211) are being isolated as a means of introducing new codons which can be used in the selection of additional missense suppressors.
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The following polynucleotides containing the antibiotic tubercidin (Tu; 4-amino-7-beta-D-ribofuranosylpyrrolo-[2,3-d]pyrimidine) were enzymatically synthesized by polymerization of adenosine 5'-diphosphate-tubercidin 5'-diphosphate mixtures with polynucleotide phosphorylase: poly(A2,Tu), poly(A,Tu2), and poly(Tu). The incorporation of the antibiotic was favored by the enzyme. The polymers are compared to poly(adenylic acid) [poly(A)] with respect to their structure, conformation, and ability to direct polylysine synthesis in a ribosome-dependent protein synthesis system. From physical data (thermal melting, NMR, and circular dichroism) it is concluded that tubercidin destabilizes the structure of the polynucleotide chain and that this may be due to an altered polarization of the nucleobases and their enhanced rotation around the N-glycosylic bond. Since there is an apparent correlation between thermal unfolding of the polymers and their ability to mediate polylysine synthesis, it is suggested that partial destacking of the messenger ribonucleic acid favors its binding to the ribosome and/or its ability to enhance codon-anticodon-specific protein synthesis.
OBJECTIVE: We hypothesized that the proteins contributing to myometrial changes during gestation could be identified indirectly by analyzing the changing pattern of messenger ribonucleic acid expression in the myometrium during pregnancy. STUDY DESIGN: Ribonucleic acid was extracted from myometrium of timed pregnant Sprague-Dawley rats on days 12, 16, 20, 21, and 22 of pregnancy and on day 1 post partum. The technique of messenger ribonucleic acid differential display, a simple and sensitive polymerase chain reaction-based method for rapidly identifying messenger ribonucleic acids whose levels increase or decrease, was performed with the nine different anchoring primers (oligodeoxythymidine11 VN: V = G, A, or C; N = G, A, or C) in combination with 24 different 10-base oligonucleotides of random sequence. The polymerase chain reaction products were separated by electrophoresis on a 5% polyacrylamide sequencing gel, and those whose levels changed were then cloned, sequenced, and compared with those in the GenBank database to determine whether they corresponded to a known sequence in the database or were novel. Semiquantitative reverse transcriptase-polymerase chain reaction was used to confirm differential expression of selected products. RESULTS: Messenger ribonucleic acid differential display revealed > 500 polymerase chain reaction products that were differentially expressed during gestation, 179 of which were cloned and sequenced. Of these, 157 were from messenger ribonucleic acids whose levels increased during gestation, and 22 were from transcripts that decreased. Eighty-seven (49%) were related to sequences in the GenBank database, of which 62 (35%) were from messenger ribonucleic acids encoding known proteins and 25 (14%) corresponded to known expressed sequence tags. The technique of semiquantitative reverse transcriptase-polymerase chain reaction confirmed the increased expression of messenger ribonucleic acids encoding beta-tropomyosin, type II phosphatidyl inositol-4-phosphate 5-kinase, and a novel myometrial messenger ribonucleic acid named RPU0901AC. CONCLUSION: Messenger ribonucleic acid differential display is a simple and sensitive method for rapidly identifying myometrial messenger ribonucleic acids that are differentially regulated during pregnancy. The identification of these differentially expressed messenger ribonucleic acids may lead to a better understanding of the molecular basis of normal and abnormal parturition.
Phenylalanine transfer ribonucleic acid from peas (Pisum sativum, Alaska) was completely digested with beef pancreatic ribonuclease (RNase I) and with ribonuclease T1. The resulting oligonucleotides were compared with those from the corresponding hydrolyses of phenylalanine transfer ribonucleic acid from wheat germ. The structures of both ribonucleic acids appeared to be identical. This report is the first to show that identical structures for the same specific acceptor transfer ribonucleic acid are present in two different plant species.
In this paper we describe the successful isolation of glyT-derived GAA suppressors. A glyT+ strain containing glyV55, the gene for a GGA/G-reading, methane sulfonate and hydroxylamine. The cells were plated to select for reversal of auxotrophy due to a trpA(GAA211) mutation. With either mutagen, greater than 85% of the prototrophs obtained were due to suppressors of the trpA mutation. Approximately 12% of the ethyl methane sulfonate-induced and 37% of the hydroxylamine-induced suppressors were shown to be about 25% cotranscucible with metB, as is glyT. The transfer ribonucleic acid from four metB-linked suppressor strains (two from each mutagen) was examined by reversed-phase column (RPC-5) chromatography. In all four cases, the glycyl-transfer ribonucleic acid profile displayed an alteration of glyT transfer ribonucleic acid. All four suppressors responded to GAG in addition to GAA but did not suppress the known mutant codons of several other trpA mutants. Other properties are discussed, along with possible reasons for our success in obtaining these suppressors.
OBJECTIVE: The aim of this study was to determine the effect of gestational age and hypoxia on the activity of ribonucleic acid polymerase in fetal guinea pig brain. STUDY DESIGN: Fetal cerebral cortical neuronal nuclei were isolated at 40, 50, and 60 days (term) of gestation to determine the effect of gestational age on the activity of ribonucleic acid polymerase I, II, and III. Pregnant guinea pigs at 60 days' gestation were randomly assigned to a normoxic or hypoxic group to determine the effect of hypoxia on ribonucleic acid polymerase activity. The fetal neuronal nuclei were pooled from 6 pregnant animals in each group. In the normoxic group the pregnant guinea pigs were exposed to room air before delivery. In the hypoxic group delivery occurred after the pregnant guinea pig had been exposed to 7% oxygen for 60 minutes. The fetuses were delivered by cesarean, and the fetal cerebral cortical neuronal nuclei were isolated immediately. Ribonucleic acid polymerase activity was determined with nuclei suspended in a buffer containing adenosine triphosphate, guanosine triphosphate, cytidine triphosphate, and tritiated uridine triphosphate. Dactinomycin (actinomycin D) and polydeoxyadenylic-thymidylic acid were used to determine the activity of bound and free ribonucleic acid polymerase. alpha-Amanitin was used to determine the activity of ribonucleic acid polymerase II. RESULTS: The activity of total (bound and free) ribonucleic acid polymerase I and III increased from 85.4 +/- 9.4 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour at 40 days' gestation to 233.3 +/- 82.1 fmol at 50 days and to 343.4 +/- 231.6 fmol at 60 days (P =.02). Total ribonucleic acid polymerase II activity increased from 19.9 +/- 6.0 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour at 40 days to 123.8 +/- 53.0 fmol at 50 days and to 200.9 +/- 77.8 fmol at 60 days (P <.01). In the term fetal guinea pig brain the activity of bound ribonucleic acid polymerase I and III decreased from 116.8 +/- 107.2 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour under normoxic conditions to 92.8 +/- 76.0 fmol in hypoxic fetal brain, a decrease of 20.5%. Free ribonucleic acid polymerase I and III activity decreased from 199.2 +/- 115.2 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour in normoxic fetal brain to 132.0 +/- 66.4 fmol in hypoxic fetal brain, a decrease of 33.8%. Free ribonucleic acid polymerase II activity decreased from 62.4 +/- 70.4 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour in normoxic fetuses to 13.6 +/- 9.6 fmol in hypoxic fetal brain, a decrease of 78.2%. In contrast, however, in term fetal guinea pig brain, bound ribonucleic acid polymerase II activity increased from 8.0 +/- 10.4 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour under normoxic conditions to 35.2 +/- 8.8 fmol in hypoxic fetal brain, an increase of 340% (P <.01). CONCLUSION: The activity of ribonucleic acid polymerases I, II, and III increases throughout the latter half of gestation, from 40 to 60 days, in the fetal guinea pig brain. Hypoxia in utero is associated with a decrease in ribonucleic acid polymerase I and III activity. Although hypoxia is associated with a decrease in free ribonucleic acid polymerase II activity, we observed a marked increase in bound ribonucleic acid polymerase II activity, which may represent a hypoxia-induced alteration of gene expression.