Distribution of lymphocyte messenger RNA during stimulation by phytohaemagglutinin

Additional of phytohaemagglutinin to cultured lymphocytes results in a progressive increase in the rate of protein synthesis of up to 7-10 times the initial rate after 20 h. Between 2 h and 12 h after the addition of mitogen, the increase in the rate of protein synthesis can be accounted for by the transfer of mRNA from messenger ribonucleoprotein complexes to polysomes. Between 12 h and 20 h the increase also reflects the accumulation of mRNA and ribosomes. However, the proportion of mRNA associated with ribosomes in unstimulated lymphocytes is unexpectedly high and paradoxically decreases during the first 2 h after mitogen addition, although the rate of protein synthesis increases. A mechanism involving mRNA selection is suggested.     

Stimulation of RNA polymerases from mouse spleen by polyamines and its modification by ammonium sulfate

Nuclear RNA polymerases Ia, Ib, II and III purified from spleen of Swiss albino mice (Mus musculus) were stimulated significantly by the polyamines, spermine and spermidine, in the presence of ammonium sulfate in vitro. In the absence of ammonium sulfate, the optimal stimulating concentrations of both the polyamines for the RNA polymerases were generally diminished and more physiological. 8 mM spermine stimulated both RNA polymerases Ia and II in the presence of sulfate ions, but inhibited both enzymes significantly in the absence of sulfate ions. Stimulation of mouse spleen RNA polymerase II by spermine affects elongation of RNA chains whereas inhibition by spermine affects initiation of RNA synthesis.     

Serum and lymphocyte activation by phytohaemagglutinin (PHA)

Activation of rat lymph-node cells in homologous serum was assessed by measuring the enhanced labelling of cells with ³H-uridine produced by PHA during a 4–6 h culture period. The degree of activation was proportional to the ratio of PHA to serum macromolecules (non-dialysable molecules) over a wide range of macromolecule concentrations. The possibility that PHA activates cells indirectly by reacting with a serum macromolecule which normally inhibits activation, was examined. No activation was produced by incubating cells in macromolecule-depleted media in the absence of PHA. Some activation was produced in macromolecule-depleted media at low PHA concentrations, but the extent of activation was only 39% of that produced in the presence of macromolecules. The results indicate that serum macromolecules both buffer cells against reaction with PHA and facilitate either the reaction of cells with PHA or the immediate cellular response to that reaction.     

Stimulation of microsomal N-demethylation by solubilized NADPH-cytochrome c reductase

The addition of solubilized NADPH-cytochrome c reductase to phenobarbital pretreated microsomes increases both the Vm value for the N-demethylation of S(+)-N, N-dimethylamphetamine and the total level of reductase activity sedimenting with microsomes. Preliminary data indicate that the increase in Vm is a nonlinear function of added reductase and demonstrates saturation at a N-demethylase level approximately five times greater than the endogenous activity. These results indicate that the added reductase is bound to microsomes and is capable of functionally coupling with the cytochrome P₄₅₀ monooxygenase system.     

Cyclic nucleotides and other factors affecting the apparent activity of RNA polymerases solubilized from rat mammary gland nuclei

• 1.1. The apparent activity (counts/min per unit enzyme) of DNA-dependent RNA polymerase (E.C. 2.7.7.6) extracted from rat mammary gland nuclei, chromatographed on DEAE-Sephadex and incubated in the presence of cyclic AMP or cyclic GMP, was altered by intentional ‘contamination’ of nuclei with cytosol, or washing them with detergent before extraction of enzyme.
• 2.2. Addition of cytosol, different column fractions or theophyllin and cyclic AMP to aliquots of enzyme could also alter the activity of the polymerase.
• 3.3. While the mechanism(s) of these effects is not established, the presence within the same column fractions of specific RNA polymerases, ³H-cyclic AMP and ³H-cyclic GMP-binding proteins and protein kinases provides a number of sites at which such putative ‘regulatory’ events could occur.
• 4.4. Results of studies using sucrose gradient centrifugation were consistent with a close association between RNA polymerase II, ³H-cyclic AMP and the calf thymus DNA template, confirming previous observations of binding by cyclic AMP to proteins contained within column fractions of RNA polymerase II.

     

DNA-dependent RNA polymerases from murine testis. Properties of two activities.

Multiple forms of DNA-dependent RNA polymerase activities have been isolated from nuclei of mouse testis. Using highly purified nuclei, two activities can be solubilized and are separable by DEAE-Sephadex chromatography; peak I eluting at 0.11–0.14 M and peak II eluting at 0.24–0.27 M (NH₄)₂SO₄. A third form of RNA polymerase activity is observed eluting at 0.31–0.33 M (NH₄)₂SO₄ when an extract from a less highly purified nuclear preparation is analysed. At concentrations of 0.125 μg/ml, peak I is insensitive to the toxin α-amanitin, peak II is totally inhibited, and peak III is partially inhibited. Peak I activity is optimal at pH 8.4 in the presence of Mg²⁺ (2–6 mM) or Mn²⁺ (1 mM) and uses native and heat-denatured DNA template equally well. Peak II has optimal activity at pH 7.9 in the presence of Mn²⁺ (2 mM) and heat-denatured DNA. Mg²⁺ has little effect on the activity of peak II.     

Reconstitution of pig lymphocyte plasma membranes from solubilized components, with particular reference to membrane-associated immunoglobulins

Plasma membranes of lymphocytes obtained from pig mesenteric lymph nodes were reconstituted after solubilization with bile salts. The proportion by weight of immunoglobulin in the reconstituted membrane was no greater than about 5-10% of that in the original membrane. Possible reasons for the low reincorporation of immunoglobulin are discussed.     

Subgenomic promoter recognition by the norovirus RNA-dependent RNA polymerases

The replication enzyme of RNA viruses must preferentially recognize their RNAs in an environment that contains an abundance of cellular RNAs. The factors responsible for specific RNA recognition are not well understood, in part because viral RNA synthesis takes place within enzyme complexes associated with modified cellular membrane compartments. Recombinant RNA-dependent RNA polymerases (RdRps) from the human norovirus and the murine norovirus (MNV) were found to preferentially recognize RNA segments that contain the promoter and a short template sequence for subgenomic RNA synthesis. Both the promoter and template sequence contribute to stable RdRp binding, accurate initiation of the subgenomic RNAs and efficient RNA synthesis. Using a method that combines RNA crosslinking and mass spectrometry, residues near the template channel of the MNV RdRp were found to contact the hairpin RNA motif. Mutations in the hairpin contact site in the MNV RdRp reduced MNV replication and virus production in cells. This work demonstrates that the specific recognition of the norovirus subgenomic promoter is through binding by the viral RdRp.