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Addition of methanol to a stringent strain of Escherichia coli, starving of methionine, stimulates unbalanced ribonucleic acid (RNA) synthesis. The newly formed RNA and ribonucleoprotein species sediment between 4S and 30S. As a result of methanol treatment, cells become permeable to actinomycin D. Damage to cellular membrane appears to influence the control for RNA synthesis.
Asporogenous mutants of Bacillus subtilis were examined for the change in template specificity of ribonucleic acid (RNA) polymerase characteristic of wild-type cells undergoing sporulation. Mutants blocked at stages II, III, and IV showed a changed specificity of the enzyme after the end of growth and were in this respect indistinguishable from the wild type. The RNA polymerase of eight stage-zero mutants (out of nine tested) which possess mutations that map at six distinct loci retained the template specificity of vegetative cells.
In an Escherichia coli rel(+)arg strain (ES-2) which carries a temperature-sensitive "G factor," the synthesis of ribonucleic acid (RNA) continues at the nonpermissive temperature (42 C) even though protein synthesis is terminated. However, at 32 C, the strain exhibits a stringent control of RNA synthesis in the absence of arginine. The stringent control of RNA synthesis imposed by trimethoprim (an inhibitor of initiation of protein synthesis) at 32 C is released at the nonpermissive temperature. Even the diauxie lag in RNA synthesis, which is regulated independently of the allelic state of the rel gene, is overcome by inactivation of the G factor. The unusual guanosine nucleotide, guanosine 5'-diphosphate 2' or 3'-diphosphate (ppGpp), is produced in small amounts during growth in strain ES-2. Withdrawal of arginine results in a greater accumulation of this compound at 32 C. At 42 C, the synthesis of ppGpp is abolished and is considerably lower than the level found in ES-2 under normal growth conditions. These results indicate that the translocation factor G plays an important role in the regulation of RNA synthesis and in the synthesis of ppGpp.
The relative amounts of two isoacceping species of tyrosine transfer ribonucleic acid, tRNATyrI and tRNATyrII, determined from reversed phase 5 profiles of tyrosyl-tRNA, prepared from Bacillus subtilis strain W168, were growth phase and medium dependent. The growth phase-dependent alterations in the relative amounts of tRNATyr species were also demonstrated in 11 asporogenous strains of B. subtilis. The proportion of tRNA-Tyr species and the extent of the alteration in their relative amounts during the transition from the exponential to the stationary phase of growth of these strains was not directly correlated with the formation of spores by strain W168 grown in various media or the stage at which the asporogenous strains are blocked in the process of sporulation.
Among 150 temperature-sensitive Saccharomyces cerevisiae mutants which we have isolated, 15 are specifically affected in ribonucleic acid (RNA) synthesis. Four of these mutants exhibit particularly drastic changes and were chosen for a more detailed study. In these four mutants, RNA synthesis is immediately blocked after a shift at the nonpermissive temperature (37 C), protein synthesis decays at a rate compatible with messenger RNA half-life, and deoxyribonucleic acid synthesis increases by about 40%. All the mutations display a recessive phenotype. The segregation of the four allelic pairs ts-/ts+ in diploids is mendelian, and the four mutants belong to three complementation groups. The elution patterns (diethylaminoethyl-Sephadex) of the three RNA polymerases of the mutants grown at 37 C for 3.5 h show very low residual activities. The in vitro thermodenaturation confirms the in vivo results; the half-lives of the mutant activities at 45 C are 10 times smaller than those of the wild-type enzymes. Polyacrylamide gel electrophoresis shows that the synthesis of all species of RNA is thermosensitive. The existence of three distinct genes, which are each indispensable for the activity of the three RNA polymerases in vivo as well as in vitro, strongly favors the hypothesis of three common subunits in the three RNA polymerases.
The synthesis of transfer ribonucleic acid (tRNA) was examined during spore formation and spore outgrowth in Bacillus subtilis by two-dimensional polyacrylamide gel electrophoresis of in vivo 32P-labeled RNA. The two-dimensional gel system separated the B. subtilis tRNA's into 32 well-resolved spots, with the relative abundances ranging from 0.9 to 17% of the total. There were several spots (five to six) resolved which were not quantitated due to their low abundance. All of the tRNA species resolved by this gel system were synthesized at every stage examined, including vegetative growth, different stages of sporulation, and different stages of outgrowth. Quantitation of the separated tRNA's showed that in general the tRNA species were present in approximately the same relative abundances at the different developmental periods. tRNA turnover and compartmentation occurring during sporulation were examined by labeling during vegetative growth followed by the addition of excess phosphate to block further 32P incorporation. The two-dimensional gels of these samples showed the same tRNA's seen during vegetative growth, and they were in approximately the same relative abundances, indicating minimal differences in the rates of turnover of individual tRNA's. Vegetatively labeled samples, chased with excess phosphate into mature spores, also showed all of the tRNA species seen during vegetative growth, but an additional five to six minor spots were also observed. These are hypothesized to arise from the loss of 3'-terminal residues from preexisting tRNA's.
The leucine auxotroph Escherichia coli 2961 exhibited stringent control of net ribonucleic acid (RNA) synthesis during amino acid starvation. After leucine was exhausted from the medium, the rate of uracil incorporation into RNA rapidly decreased to 2 to 4% of the prestarvation value. Infection of the starved cells with T2 phage stimulated uracil incorporation to a level equivalent to that of unstarved, T2-infected cells. The RNA synthesized during leucine starvation of the T2-infected cells consisted of T2 and E. coli messenger RNA, but stable ribosomal RNA (23S and 16S) did not appear to be synthesized. It is concluded that one or more T2-specific proteins are required to shut off host messenger RNA synthesis. Furthermore, transcription of E. coli and T2 deoxyribonucleic acid is not necessarily coupled to the translation of messenger RNA during stringent control of net RNA synthesis.
"L-cell virion," obtained from chronically infected 929 L-cell lines, was purified on 70% sucrose pads and its ribonucleic acid (RNA) was extracted and analyzed on sucrose gradients. Two RNA components were isolated from the virion, a rapidly sedimenting species (82S) and a more slowly sedimenting species (6S). When the fast-moving component was heated to 45 C, it yielded a homogeneous peak which now sedimented at approximately 72S. Upon heating to 70 C, the peak dissociates into a smaller homogeneous RNA species with a sedimentation coefficient of approximately 33S. Ribonuclease treatment and base composition analysis revealed that the fast-moving species is a single-stranded molecule. Base ratio analysis also revealed that the small (6S) RNA component is similar in its base composition to L-cell ribosomal RNA and distinctly different from the base composition of the large (72S) RNA component. Actinomycin D inhibits production of the L-cell virion by the L-cell line.
Evidence is presented that, in frog virus 3-infected BHK cells, inhibition of ribonucleic acid (RNA) synthesis is paralleled by a decreased activity of host cell RNA polymerase II, while the template ability of host cell nuclei and chromatin is unaffected.
The expression of GHRH receptor (GHRH-R) messenger ribonucleic acid (mRNA) was studied in 22 pituitary adenomas and 2 normal anterior pituitaries. Northern blot analysis revealed that GHRH-R mRNA were expressed in all 14 GH-producing adenomas, 1 of 3 ACTH-producing adenomas, the 1 PRL-producing adenoma, 2 of 4 nonfunctioning adenomas, and the 2 normal anterior pituitaries. Their expression levels varied among GH-producing adenomas and were relatively low in GH-nonproducing adenomas. In addition to the major transcript with a molecular mass of 2.0 kilobases (kb), the transcripts were identified at 2.8 and 4.5 kb in some GH-producing adenomas. To examine the structural variations in GHRH-R mRNA in pituitary adenomas, we amplified the complementary DNA fragment encompassing the region from the third cytoplasmic loop to the sixth transmembrane domain of GHRH-R. This region was selected because this region of the G protein-coupled receptor has been known to interact with G protein. Two amplified fragments with the molecular masses of 250 and 810 base pairs were identified by the reverse transcriptase-polymerase chain reaction method. The nucleotide sequence of a smaller fragment, which was the expected size, revealed that no mutations were found in this region in 10 GH-producing adenomas examined. However, a larger fragment contained the currently unidentified insertion. Compared with the genomic DNA sequence, this insertion was found to be generated through alternative splicing. In addition, this variant form contained the premature stop codon in-frame, indicating that it encodes the truncated GHRH-R. This insertion-specific probe could hybridize with 2.8- and 4.5-kb species of GHRH-R mRNA on Northern blot analysis, and these transcripts were expressed mainly in GH-producing adenomas. Finally, study of cell transfection and cAMP measurement revealed that this truncated GHRH-R was unable to transmit GHRH signals. These results suggest that some GH-producing adenomas preferentially express the truncated GHRH-R as a nonfunctioning receptor through alternative splicing.
Inhibin alpha and beta A subunit messenger ribonucleic acid (mRNA) levels were measured quantitatively by reverse transcription-polymerase chain reaction (RT-PCR) in human pituitary adenomas. The inhibin alpha subunit mRNA levels were undetectably low in cultured adenoma tissues, but beta A mRNA were 0.383 +/- 0.074 in 3 GH adenomas, 0.672 +/- 0.140 in 3 prolactinomas and 0.957 +/- 0.414 molecules/cell in 3 non-functioning adenomas. The addition of 10(-8) M activin A decreased the beta A mRNA levels within 4 h in 1 of 3 GH adenomas, 2 of 3 prolactinomas and 2 of 3 non-functioning adenomas, though the decreases were not statistically significant. The results showed an abundance of beta A subunit mRNA compared with alpha subunit mRNA in all human pituitary adenomas and a local role for activin in its own production through inhibin beta A mRNA subunit expression.
Messenger ribonucleic acid (mRNA) levels and activities of renal 25-hydroxyvitamin D3-24-hydroxylase (24-OHase) were determined in 3 groups of rats: vitamin D-deficient, normal, and 1 alpha-hydroxyvitamin D3 (1 alpha OHD3)-administered rats. To measure renal 24-OHase mRNA, a DNA probe complementary to the reported sequence of the recently cloned P450 component was employed. The DNA probe hybridized with 24-OHase mRNA. Northern blot analysis indicated that the size of the transcript was approximately 3.4 kb, which was similar in size to that of the previous report. Oral administration of 1 alpha OHD3 (2 micrograms/kg bw/day x 7 days) markedly increased the plasma concentration of 1,25-dihydroxyvitamin D (1,25(OH)2D), renal 24-OHase activity and the renal 24-OHase mRNA level. However, the levels of 24-OHase mRNA were undetectable in vitamin D-deficient and normal rat kidneys, while a significant amount of 24-OHase activity was observed in the normal rat kidney. In vitamin D-deficient rats, renal 25-hydroxyvitamin D3-1 alpha-hydroxylase activity was markedly elevated and 24-OHase activity was completely abolished. When an oligonucleotide probe complementary to coding sequence of heme binding region was used for dot blot analysis, the mRNA was clearly detected in normal rat kidney. Furthermore, activity of the enzyme was attenuated by human parathyroid hormone (1-34) but the mRNA abundance did not change with 2 nmol (every 4 h, 5 times) of hormone treatment in 1 alpha OHD3-dosed thyroparathyroidectomized rats. The present study demonstrates that induction of 24-OHase activity by 1,25(OH)2D3 occurs at least in large part through increase of the gene expression in the kidney. Furthermore, these findings suggest that 2 forms (inducible and constitutive) of 24-OHase exist in rat kidney, and that the previously cloned P450 component is the inducible form of 24-OHase.
Deviations of the enzyme activity, immunoreactivity and messenger ribonucleic acid (mRNA) levels of glutathione peroxidase (GSH-PO) in 3'-methyl-4-dimethylaminoazobenzene- induced hepatocellular carcinoma of the rat were investigated. Enzyme activities of GSH-PO were significantly lower in hepatocellular carcinomas than those in the normal control rat liver. Immunohistochemically, GSH-PO was strongly localized in normal hepatocytes, but was only faintly stained in hepatocellular carcinoma cells. Heterogeneous staining patterns of GSH-PO were observed among individual cancer cells. In Northern blot analysis, GSH-PO mRNA in the cancer tissue was decreased to two thirds of the level in normal hepatocytes. It was suggested that suppressed expression of GSH-PO in carcinogen-induced hepatocellular carcinomas occurred at the level of mRNA transcription.
The effects of hypothyroidism induced by propylthiouracil (PTU) treatment on growth hormone (GH) and prolactin (PRL) messenger ribonucleic acid (mRNA) levels were analyzed in adult female rat adenohypophyses by in situ hybridization histochemistry and Northern hybridization analyses. Twenty-eight days of PTU treatment produced a significant decrease in GH mRNA levels and a smaller decrease in PRL mRNA determined by both in situ hybridization histochemistry and Northern hybridization analyses. A combined procedure of in situ hybridization histochemistry followed by immunochemistry on the same sections revealed mammosomatotropic cells expressing GH mRNA and PRL protein in the same pituitary cells from all treatment groups. Cells expressing GH mRNA and thyroid-stimulating hormone protein were not detected by this method. Immunochemical staining revealed a decrease in GH cells and an increase in thyroid-stimulating hormone cells in hypothyroid rats. Cells expressing both GH and thyroid-stimulating hormone protein were not detected by immunostaining. These results indicate that hypothyroidism produces significant decreases in GH mRNA and also decreases PRL mRNA and that mammosomatotropic cells can be detected in pituitaries from normal and hypothyroid rats.
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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-week-old chickens and used to raise a specific antiserum in rabbits. This antiserum was coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA. RNA was isolated from identical breast muscles and consecutively fractionated with several techniques to yield a preparation of GAPDH mRNA which was at least 50% pure. Double-stranded cDNA was made against this purified RNA, inserted into pBR322, and used to transform Escherichia coli. Recombinants were screened by colony filter hybridization with a cDNA probe made against the purified RNA. The hybridization-positive clone with the largest insert, pGAD-28, was then characterized by using pGAD-28-cellulose to select complementary RNA from total poly(A) RNA and then translating the hybridization-selected RNA in vitro. The single translation product was shown to be GAPDH by (1) comigration with pure GAPDH on sodium dodecyl sulfate-polyacrylamide gels, (2) precipitation with specific anti-GAPDH antiserum, (3) cyanylation fingerprinting, and (4) AMP-agarose affinity chromatography. pGAD-28 was mapped with several restriction enzymes and then sequenced by the method of Maxam and Gilbert [Maxam, A. M., & Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560]. The 1261-nucleotide insert was found to contain 29 nucleotides of noncoding sequence at the 5' end, the entire coding region, and 230 nucleotides of the 3'-noncoding region including a poly(A) addition signal (AATAAA) and the first five residues of the poly(A) tail.
The horseradish peroxidase catalyzed aerobic oxidation of the auxin indole-3-acetic acid generates triplet indole-3-aldehyde in high yield. The excited species is quenched by oxygen with formation of singlet oxygen, which is responsible for the observed photon emission and can be trapped by suitable agents. tRNA dramatically enhances the emission as a result of energy transfer from triplet indole-3-aldehyde to a 4-thiouridine group in tRNA. Triplet indole-3-aldehyde also adds covalently to tRNA. The results provide a possible mechanism for the auxin-tRNA interaction in vivo.
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