Effect of incubation conditions, inhibitors, 2-mercaptoethanol and testosterone on RNA synthesis in ram spermatozoa

In vitro studies on RNA synthesis using washed ram spermatozoa were carried out by measuring the incorporation of (3)H-uridine into RNA. Penicillin-G (100 mug/ml medium) was added to prevent contamination by microorganisms. Spermatozoa were quickly separated from seminal plasma by washing twice in Tris-HCl buffer (at pH 7.2) and centrifuged at 1,000 g for 5 min. Washed spermatozoa were then diluted to 1 10 , 1 20 or 1 40 (v/v) by the same buffer system (containing 400 mg% glucose) and were incubated in air at 37 degrees C for 1, 2 and 4 h. Results indicated that the rate of RNA synthesis was maximal at 1 40 semenbuffer dilution (5-8 x 10(7) spermatozoa/ml) and increased linearly up to 4 h of incubation. The rate of RNA synthesis at 1 40 dilution also increased linearly as the dose of exogenous glucose substrate was increased up to 400 mg%. Denaturation of the ram spermatozoa by 1% HgCl(2) caused almost complete inhibition of RNA synthesis that amounted to 97% of the control samples. Incubation of spermatozoa with 50, 100 or 200 mug/ml chloramphenicol also inhibited uridine incorporation by 86 to 94%, while equivalent doses of cycloheximide did not. On the other hand, the incorporation of (3)H-uridine into the RNA of ram spermatozoa was significantly enhanced by graded doses of 2-mercaptoethanol (0.2, 0.4 and 0.8 muM) and of testosterone (15 and 30 mug/ml). The results of this study indicate RNA synthesis, mainly of mitochondrial origin, by mature ram sperm. The data also suggest a role for intracellular cyclic adenosine monophosphate in the regulation of sperm RNA synthesis.     

Role of ribonucleic acid synthesis in conjugational transfer of chromosomal and plasmid deoxyribonucleic acids

A strain of Escherichia coli K-12 containing mutations that allow for the experimental control of RNA and DNA syntheses was constructed to investigate the role that RNA synthesis plays in conjugational DNA transfer when DNA replication is inhibited. The mutations possessed by this strain and its donor derivatives include: (i) thyA, which blocks synthesis of dTMP, causing a requirement for thymine; (ii) deoC, which blocks breakdown of deoxyribose 5-phosphate, permitting growth with low levels of thymine; (iii) pyrF, which blocks synthesis of UMP from OMP, imposing a requirement for uridine; (iv) cdd and pyrG, which block the deamination of cytidine to uridine and the synthesis of CTP from UTP, respectively, causing a requirement for cytidine; (v) codA and codB, which block the deamination of cytosine to uracil and cytosine transport, respectively, preventing the substitution of cytosine for cytidine; and (vi) dnaB, which blocks vegetative but not conjugational DNA replication at 42 degrees C. DNA synthesis can be blocked in the donor strains by the addition of excess uridine when exogenous thymine is not present. We found that RNA synthesis can also be blocked by addition of excess uridine when exogenous cytidine is not present. Blocking RNA synthesis prior to mating, under conditions in which DNA synthesis either is or is not inhibited, depresses DNA transfer. However, under conditions in which DNA synthesis is inhibited, the blocking of RNA synthesis immediately after mating has commenced had no effect on continued conjugational transfer of DNA. Thus, RNA synthesis is needed to initiate but not to continue conjugational DNA transfer.     

Bentazon influence on selected metabolic processes of isolated bean leaf cells

Time- and concentration-course studies were conducted to determine the effect of bentazon [3-isopropyl-1H-2,l,3,-benzothiadiazin-4(3H)-one 2,2-dioxide] on photosynthesis, RNA synthesis, protein synthesis, and lipid synthesis using enzymatically isolated leaf cells of red kidney bean (Phaseolus vulgaris L.). Photosynthesis and RNA synthesis were inhibited about 75% at 1 M bentazon at the 30 min treatment period. This was the lowest concentration and shortest time that significantly inhibited any of these four processes. The degree of inhibition of photosynthesis was greater than the degree of inhibition of RNA synthesis at higher concentrations and/or longer time periods. At 10 M bentazon, protein synthesis and lipid synthesis were also inhibited. Lipid synthesis was stimulated at 0.1 and 1 M at 120 min.     

Non-coordinate synthesis of RNA polymerase beta beta'' subunits in a temperature-sensitive beta''-subunit mutant of Escherichia coli

Summary On exposure to high temperature of a temperature-sensitive RNA polymerase subunit (rpoC92) mutant of Escherichia coli, selective reduction was observed in the rate of synthesis of a group of proteins including RNA polymerase subunit. The finding that the synthesis of subunit but not subunit was specifically repressed in this mutant grown at non-permissive temperature indicates that the functionally intact RNA polymerase is required for the synthesis of subunits be coordinated. In addition, the assembly of newly synthesized RNA polymerase subunits was inefficient in this mutant at the steps where altered subunit was involved, and the unassembled enzyme subunits were rapidly and preferentially degraded. During recovery to non-restricted growth, the synthesis of both and subunits was transiently enhanced in parallel leading to recovery of the intracellular concentration of functional RNA polymerase.     

Butachlor influence on selected metabolic processes of plant cells and tissues

Time- and concentration-course studies were conducted to determine the effects of butachlor (N-[butoxymethyl]-2-chloro-2,6-diethylacetanilide) on photosynthesis, protein synthesis, RNA synthesis, and lipid synthesis using isolated leaf cells of red kidney bean (Phaseolus vulgaris L.). At the 2-h incubation period, butachlor inhibited photosynthesis, protein synthesis, RNA synthesis, and lipid synthesis 99, 99, 96, and 81% respectively at 100 M, and 0, 19, 17, and 40% respectively at 10 M. At 100 M and 15-, 30-, and 60-min incubations, RNA synthesis was inhibited 20, 76 and 90% respectively, and lipid synthesis 35, 48, and 62% respectively; photosynthesis and protein synthesis were inhibited over 90% at all of these time periods. The effects of 50 M butachlor on protein and RNA synthesis in rice (Oryza sativa L.) and barnyardgrass (Echinochloa crusgalli L.) root and shoot segments were also investigated. Protein synthesis was inhibited in both species and to a greater degree in roots (81–90%) than in shoots (55–65%). RNA synthesis was inhibited 33% in barn-yardgrass roots but not significantly in barnyardgrass shoots or either organ of rice.     

On the adjustment of the sex ratio and the gregarious behavior of animal populations.

Ribosome synthesis in bacteria is linked to RNA polymerase synthesis; both synthesis rates depend upon the values of six parameters: (1) fraction of total ribosomes that is functioning, (2) fraction of total RNA polymerase that is functioning, (3) fraction of functioning RNA polymerase engaged in rRNA synthesis, (4) fraction of total protein that is RNA polymerase protein, (5) peptide chain elongation rate, (6) rRNA chain elongation rate. If these parameters are constant in time, then the numbers of both ribosomes and RNA polymerase molecules increase exponentially. It is shown how the rate constant (fractional increase per unit of time) relates to these parameters and how the kinetics of ribosome and RNA polymerase synthesis respond to a change in any of these parameters.     

Inhibition by trioxalen (psoralen) plus near-ultraviolet light of the induction of ornithine decarboxylase in Chinese-hamster cells.

Trioxalen (trimethylpsoralen) plus near-u.v. light, a potent inhibitor of DNA and RNA synthesis, inhibits the induction of ornithine decarboxylase in stationary-phase V79 fibroblasts. It does not affect the translation of pre-existing mRNA. The method, in view of its high degree of specificity and precise timing, is a better choice for inhibiting RNA synthesis than the commonly used chemical inhibitors and precursor analogues.     

Reciprocal post-translational regulation of renal 1 alpha- and 24-hydroxylases of 25-hydroxyvitamin D3 by phosphorylation of ferredoxin. mRNA-directed cell-free synthesis and immunoisolation of ferredoxin.

We have used a cell-free rabbit reticulocyte translational system programmed with polyadenylated [poly(A)+] RNA prepared from chick kidney tissue to study the synthesis of nascent ferredoxin, a class of iron-sulphur-containing proteins functional in the renal mitochondrial 1 alpha- and 24-hydroxylases of 25-hydroxyvitamin D3. The synthesis of ferredoxin was monitored by determining [35S]methionine incorporation into ferredoxin and quantified by SDS/PAGE and autoradiography after immunoprecipitation from the total translation products. Compared with normal controls, vitamin D deprivation caused a significant increase in the net synthesis of nascent ferredoxin with an Mr of 12,000-13,000. [3H]Orotate incorporation as uridine into kidney poly(A)+ RNA was stimulated by aminophylline, a potent inducer of 25-hydroxyvitamin D3 24-hydroxylase; however, the amount of nascent ferredoxin synthesis was the same as in normal controls. Also, partially purified chick kidney mitochondrial cyclic AMP-stimulated protein kinase catalysed the phosphorylation of ferredoxin in vitro. The catalytic activity of the ferredoxin in 1 alpha- and 24-hydroxylations of 25-hydroxyvitamin D3 in reconstituted systems consisting of cytochrome P-450 and ferredoxin reductase was altered with ferredoxin phosphorylation. The phosphorylation caused inhibition of the 1 alpha-hydroxylase activity while at the same time it stimulated the 24-hydroxylase. Authentic 1 alpha,25- and 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were used as standards to monitor the separation of the enzymic products by h.p.l.c. using methanol/water (4:1, v/v) as solvent. These results indicate that, in the absence of vitamin D or its metabolites in the deficient state, the synthesis of ferredoxin necessary for the 1 alpha-hydroxylase is accentuated, whereas the stimulation of the 24-hydroxylase requires the phosphorylation of existing ferredoxin without a net gain in its synthesis. This would suggest a post-translational regulation of the 1 alpha- and 24-hydroxylases. A model delineating the various aspects of this study is presented.     

Requirements at the 3' end of the sindbis virus genome for efficient synthesis of minus-strand RNA

The 3'-untranslated region of the Sindbis virus genome is 0.3 kb in length with a 19-nucleotide conserved sequence element (3' CSE) immediately preceding the 3'-poly(A) tail. The 3' CSE and poly(A) tail have been assumed to constitute the core promoter for minus-strand RNA synthesis during genome replication; however, their involvement in this process has not been formally demonstrated. Utilizing both in vitro and in vivo analyses, we have examined the role of these elements in the initiation of minus-strand RNA synthesis. The major findings of this study with regard to efficient minus-strand RNA synthesis are the following: (i) the wild-type 3' CSE and the poly(A) tail are required, (ii) the poly(A) tail must be a minimum of 11 to 12 residues in length and immediately follow the 3' CSE, (iii) deletion or substitution of the 3' 13 nucleotides of the 3' CSE severely inhibits minus-strand RNA synthesis, (iv) templates possessing non-wild-type 3' sequences previously demonstrated to support virus replication do not program efficient RNA synthesis, and (v) insertion of uridylate residues between the poly(A) tail and a non-wild-type 3' sequence can restore promoter function to a limited extent. This study shows that the optimal structure of the 3' component of the minus-strand promoter is the wild-type 3' CSE followed a poly(A) tail of at least 11 residues. Our findings also show that insertion of nontemplated bases can restore function to an inactive promoter.     

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.     

A Method for the Selective Removal of Deoxyribonucleic Acid from Tissue Sections

The deoxyrihonucleic acid (DNA) of chromatin undergoar depurinization on mild acid hydrolysis with a picric acid-formaldehyde mixture (Bouin's fluid). The apurinic acid thus formed is degraded by condensation with aniline and is lost from tissue sections, but ribonucleic acid (RNA) in nucleoli and cytoplasm is well preserved. Technique: Fi in Carnoy's fluid (ethanol:acetic acid 3:1 or ethanol:chloroform:acetic acid 6:3:1) or in aldehydes (10% formalin or 2.5% glutaraldehyde bsered to pH 7.0). Hydrolyse deparaEnii sections 12-24 hr at 27-50 C in Bouin's fluid, wash in distilled water, immerse in 25% (v/v) acetic acid, treat 1 hr at 27-30 C with 10% (v/v) dine in 25% acetic acid, wash in 25% acetic acid and then in water. Stain 10-40 min with 0₃% toluidine blue in 0.05 M potassium biphthalate bder (pH 4.0); rinse in distilled water, pass to 10% (w/v) ammonium molybdate for 1 min, rinse again in water and pass through tert-butanol and xylene to a synthetic resin. Chromatin and chromosomes are pale green; RNA in nucleoli and cytoplasm deep purple.     

Mutants in the y region of bacteriophage lambda constitutive for repressor synthesis: their isolation and the characterization of the Hyp phenotype

Bacteriophage lambdahyp mutants have been isolated as survivors of Escherichia coli K-12 bacteria lysogenic for lambda Nam7am53cI857. The hyp mutants are characterized by (i) their localization in the y region very close to the imm lambda/imm434 boundary, (ii) polarity on O gene expression, (iii) immediate recovery of lambda immunity at 30 degrees C after prolonged growth of lambda Nam7am53cI857 hyp lysogens at 42 degrees C even in the presence of an active cro gene product, (iv) ability of phage lambda v2v3vs326 but not lambda v1v2v3 to propagate on lambda cI+hyp lysogens, (v) inability to express lambda exonuclease activity after prophage induction, and (vi) inviability at any temperature of phage carrying the hyp mutation. All these properties are referred to collectively as the Hyp phenotype. We show that the Hyp phenotype is due to cII-independent constitutive cI-gene-product synthesis originating in the y region, which results in the synthesis of anti-cro RNA species, and constitutive levels of cro gene product present even in lambda cI+hyp lysogens. A model is presented which is consistent with all the experimental observations.     

Effect of cyclic AMP, imidazole and triiodothyronine on the rate of RNA synthesis by ejaculated ram spermatozoa

The effects of cyclic AMP (cAMP) and of triiodothyronine (T(3)) on the enhancement of sperm motility and metabolism are well documented, and the present study was undertaken to investigate the mechanisms underlying these effects in terms of their influence on sperm RNA synthesis in vitro. Washed ram sperm were diluted to 1 40 (v/v) with incubation buffer that contained 100 mug/ml penicillin-G and 400 mg% glucose, followed by incubation at 37 degrees C for a period of 4 h. Washed ram spermatozoa incubated with graded doses of cAMP (10(-8), 10(-6) and 10(-4) M) showed significant enhancements of the rate of (3)H-uridine incorporation into RNA, with maximal effect occurring at 10(-8) M. The presence of 3.75, 7.50 or 15.00 muM T(3) also stimulated the rate of RNA synthesis by the washed ram sperm, with maximal effect occurring at 7.50 muM. On the contrary, imidazole (a compound known to stimulate phosphodiesterase activity and consequently to decrease the intracellular cAMP levels in many tissues) was found to cause consistent inhibition of spermatozoal RNA synthesis. The inhibition was 47, 90 and 92% of control for 10, 50 and 100 mM imidazole, respectively. The results obtained indicate that cAMP may act either as a first or a second messenger in the mature sperm. The data also indicate that T(3) (possibly mediated by cAMP) may act on the ram sperm by the induction of enzymes, which are required for the well-known effects of this hormone in enhancing the sperm metabolic activity.     

The effect of tri-iodothyronine administration on protein synthesis in the diabetic rat.

In young male rats diabetes caused decreases in circulating free tri-iodothyronine and RNA concentration in liver and muscle, and in the rate of protein synthesis per unit of RNA (RNA activity) in muscle. Tri-iodothyronine treatment significantly increased RNA concentrations, but not RNA activity, in these tissues. Thus: (1) impaired thyroid status is a component of the diabetic condition; (2) tri-iodothyronine cannot stimulate the translational phase of protein synthesis in the diabetic rat.     

Relationship between RNA synthesis and the Ca2+-filled state of the nuclear envelope store

RNA synthesis and ATP-dependent (45)Ca(2+) uptake were measured simultaneously in isolated nuclear fraction of rat liver nuclei. Maximal level of RNA synthesis was obtained under ATP-dependent (45)Ca(2+)-uptake conditions (1 microM free [Ca(2+)] and 1 mM ATP in the bathing solution). This experimental condition was defined as "stimulated nuclei" condition. ATP-dependent (45)Ca(2+) uptake was inhibited using different strategies including: (a) eliminating Ca(2+) (1 mM EGTA); (b) lowering the ATP concentration; (c) modifying nuclear envelope membranes Ca(2+) permeability (Ca(2+) ionophores); or (d) inhibiting the nuclear Ca(2+) pump (thapsigargin and 3',3',5',5'-tetraiodophenolsulfonephthalein). Under all the above conditions, RNA synthesis was lower than in "stimulated nuclei" condition. In the presence of ionomycin, RNA synthesis was significantly higher at 500 nM free [Ca(2+)], as compared with RNA synthesis in a Ca(2+)-free medium or at 1muM free [Ca(2+)]. However, even in such condition (500 nM free [Ca(2+)]), RNA synthesis was lower than RNA synthesis obtained in "stimulated nuclei" condition. We suggest two components for the effect of Ca(2+) on RNA synthesis: (A) a direct effect of nucleoplasmic [Ca(2+)]; and (B) an effect dependent on the accumulation of Ca(2+) in the nuclear envelope store mediated by the SERCA nuclear Ca(2+) pump.