Selective alterations in hepatic enzyme response after reduction of nuclear triiodothyronine receptor sites by partial hepatectomy and starvation.

Barley embryo 5S rRNA hybridizes efficiently with barley embryo 18S rRNA but not with 26S rRNA. Mouse sarcoma 5S rRNA also selectively hybridizes, to a smaller extent, with mouse sarcoma 18S rRNA. The barley embryo 5S–18S rRNA complex has a sharp melting profile and a “Tm” of $\text{ca}$. 59° in 0.1 $\text{M}$ NaCl. The mouse sarcoma 5S–18S rRNA complex has a broader transition breadth and a “Tm” of $\text{ca}$ 52°. The conditions used for hybridization lead to very specific reconstitution of the “natural” complex between 5.8S and 26S–28S rRNA since both the $\text{in}\text{vivo}\text{and}\text{in}\text{vitro}$ complexes between 5.8S and 26S–28S rRNA from barley embryos and mouse sarcoma have equally sharp melting profiles and a “Tm” of $\text{ca}$. 52° in 0.1 $\text{M}$ NaCl.     

Escherichiacoli mutants deficient in exoribonucleases

Strain S296, isolated by screening 2000 colonies after nitrosoguanidine mutagenesis, yields extracts with less than 1% of wild-type RNase activity against (³H) poly(U). Unlike other $\text{E.}\text{coli}$ strains, S296 grows with a doubling time of about 2 hr., both in nutrient broth and in minimal medium, and at 30°, 37° and 42°. The strain retains 10 to 20% of wild-type exonuclease activity against (³H) rRNA or T4 phage-specific mRNA; but two further mutants, made by screening mutagenized colonies of strain S296, are reduced to 3% of wild-type activity against those substrates as well.     

Hydrodynamic properties of 5.8S rRNA,salt and temperature effects

Sedimentation coefficients and apparent molecular masses of 5.8S rRNA from rat liver and yeast (Saccharomyces cerevisiae) depend considerably on the ionic strength and the kind of ions in solution. At 20°C the sedimentation coefficient of 5.8S rRNA in 10 mm sodium cacodylate, pH 7.0, amounts to 5.1 ± 0.2 S. By addition of NaCl up to 1.1 m the data increase reversibly to 6.1 ± 0.2 S (rat liver) or 5.4 ± 0.1 S (yeast) without significant changes of the molar mass (52 000 ± 2000) g/mol. Similar effects but with different extent were obtained using KCl or LiCl. These results can be explained by counterion effects on the conformation and changing of the water shell surrounding the RNA molecule. Short heat incubation (5 min at 65°C) and immediate cooling of rat liver 5.8S rRNA lead to dimer or oligomer formation. Its portions depend strongly on RNA concentration and are enhanced also with increasing NaCl concentration and incubation temperature as can be seen fro higher sedimentation coefficients and molecular masses as well as from additional bands in the electrophoretic pattern. At 20°C MgCl₂ provokes, in concentrations up to 1.5 mm, a reversible increase of sedimentation coefficients of rat liver 5.8S rRNA to 6.65 ± 0.1 S whereas the molecular mass remains unchanged indicating strong Mg⁺⁺ effects on conformation and/or water shell of the 5.8S rRNA. A further increase of sedimentation coefficients up to 8.2 ± 0.1 S combined with higher apparent molar masses up to 90 000 g/mol was observed in the presence of 30 to 50 mm MgCl₂. In this concentration range of Mg⁺⁺ the association constants of 5.8S rRNA dimerization increase from about $\text{10}{}^5\text{to}\text{3 × 10}{}^7\text{m}{}^{\mathrm{?1}}$. After removal of free Mg⁺⁺ by addition of EDTA the 5.8S rRNA dimers dissociate if no incubation step at higher temperature in involved. The Mg⁺⁺ induced 5.8S rRNA dimers differ in their stability from those formed by incubation at 65°C in the presence of higher concentrations of monovalent ions.     

Destabilization of the secondary structure of RNA by ribosomal protein S1 from escherichia coli

S1 is an acidic protein associated with the 3′ end of 16S RNA; it is indispensable for ribosomal binding of natural mRNA. We find that S1 unfolds single stranded stacked or helical polynucleotides (poly rA, poly rC, poly rU). It prevents the formation of poly (rA + rU) and poly (rI + rC) duplexes at 10–25 mM NaCl but not at 50–100 mM NaCl. Partial, salt reversible denaturation is also seen with coliphage MS2 RNA, $\text{E. coli}$ rRNA and tRNA. Generally, only duplex structures with a Tm greater than about 55° are formed in the presence of S1. The protein unfolds single stranded DNA but not poly d(A·T).     

Synthesis and characterization of a DNA probe complementary to rat liver 28S ribosomal RNA.

Conditions for the production of a complementary DNA sequence for use in studies of ribosomal RNA are described. $\text{E}$. $\text{coli}$ DNA polymerase I is used to transcribe highly purified 28S ribosomal RNA from rat liver. The reaction is sensitive to the tertiary structure of the rRNA template-primer. The complementary DNA hybridizes to its rRNA template with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.02. The hybrid formed between 28S ribosomal RNA and complementary DNA has a T_m of 73°C. The probe reacts with total rat nuclear RNA with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.0.     

A mutation in Escherichia coli that mimics diauxie lag.

We have described a mutant of $\text{E.}\text{coli}$ (2S142) which shows a specific inhibition of stable RNA synthesis at 42°. The temperature sensitive lesion differs from the stringent response to amino acid starvation in that the shut off of rRNA synthesis is not associated with an inhibition of protein synthesis. The decay of ppGpp is slow at 42° with little or no pppGpp detectable. This slow decay rate is not observed in the parental strain, D10, or in 2S142 at 30°. Neither 2S142 or D10 are spoT, nor does the temperature sensitive lesion map near the spoT locus. Thus, the effect of the temperature sensitive lesion on ppGpp metabolism and rRNA synthesis seems to resemble a carbon source downshift (diauxie lag) rather than a stringent response to amino acid starvation.     

Binding of steroids to P.testosteroniΔ5-3-ketosteroid isomerase: Measurement of the number of binding sites by equilibrium dialysis

The binding of progesterone, 17β-estradiol and 19-nortestosterone acetate to the Δ⁵-3-ketosteroid isomerase from $\text{Pseudomonas}$$\text{testosteroni}$ has been investigated by the technique of equilibrium dialysis. Under the conditions used, all three steroids formed 2:1 complexes with each molecule of enzyme dimer (M.W. = 26,788). No evidence of any cooperative binding phenomena was obtained. The dissociation constants of the enzyme steroid complexes at 25°C were: progesterone, 2.2 μ$\text{M}$; estradiol, 2.5 μ$\text{M}$; 19-nortestosterone acetate, 9.2 μ$\text{M}$.     

Translation of mouse interferon mRNA in Xenopus laevis oocytes and in rabbit reticulocyte lysates

Mouse interferon mRNA, extracted from NDV (Newcastle disease virus)-induced L-929 cells has been translated with high efficiency in $\text{Xenopus laevis}$ oocytes and rabbit reticulocyte lysates. The translational efficiency of a crude RNA extract was 10 640 interferon units/mg RNA/hour for the $\text{Xenopus}$ oocytes and 4 012 interferon units/mg RNA/hour for the reticulocyte lysates. The translation product fulfilled the usual criteria for mouse interferon, $\text{viz.}$ species specificity and neutralization by specific anti-mouse interferon antiserum. Upon injection of crude interferon mRNA into $\text{Xenopus}$ oocytes, interferon activity appeared both in the oocyte homogenates and the oocyte incubation medium. When analyzed by velocity sedimentation in formamidesucrose, the mouse interferon mRNA showed a rather sharp peak halfway between the 4 S and 18 S RNA markers, as could be expected from a mRNA which codes for a 20,000 dalton protein.     

Cross-linking studies on a cytochrome c-cytochrome c oxidase complex.

A cytochrome $\text{c}$ - cytochrome $\text{c}$ oxidase complex containing 0.8–1.0 moles of cytochrome $\text{c}$ per mole of cytochrome $\text{c}$ oxidase (heme a + a₃) was isolated as described by Ferguson-Miller, S., Brautigan, D.L., and Margoliash E., J. Biol. Chem. $\text{251}$, 1104 (1976). This complex was reacted with dithiobissuccinimidyl propionate, an 11 Å bridging bifunctional reagent, and the cross-linked products obtained were analyzed by two dimensional gel electrophoresis. Cytochrome $\text{c}$ was cross-linked to subunit II of cytochrome $\text{c}$ oxidase. Other cross-linked products were formed involving different subunits of cytochrome $\text{c}$ oxidase. These included I+V, II+V, III+V, V+VII, IV+VI and IV+VII. Experiments are also described using N,N′-bis(3-succinimidyloxycarbonylpropyl) tartarate. The major product formed with this 18 Å bridging bifunctional reagent was a pair containing II+VI.     

alpha-Fetoprotein biosynthesis and hepatocellular differentiation

In anemia of the Belgrade rat ($\text{b}\text{b}$) reticulocytes contain less than half of the normal amount of mRNA for seven adult rat globin chains. cDNA hybridization measurements of the relative sizes of polysomal and nonpolysomal pools of globin mRNAs in these cells show that 45% of all globin mRNA molecules are not used at any given time in protein synthesis. This implies a translational control which ensures a production of globin chains in a correct ratio despite a severe mRNA unbalance.     

A novel, inexpensive, and sensitive method for analysis of tyrosine hydroxylase activity in tissue samples.

Tyrosine hydroxylase activity in whole mouse brains was measured $\text{in v}\text{vitro}$. The L-dihydroxyphenylaline (L-DOPA) formed by the enzyme was quantitated by liquid chromatography with electrochemical detection (LCEC). An investigation of the incubation factors (added Fe⁺², DOPA decarboxylase inhibitor concentration, substrate concentration, amount of tissue, time of incubation) is reported. Under optimal conditions the activity was found to be 15.1 ± 0.6 (S.E.M.) nmol DOPA formed/hr./g. tissue.     

Mycoplasma pneumoniae: A prokaryote which consumes oxygen and generates superoxide but which lacks superoxide dismutase

Superoxide dismutase and catalase were not detected in $\text{M}$. $\text{pneumoniae}$ and several other species of $\text{Mycoplasma}$ some of which consume oxygen and secrete H₂O₂. $\text{M}$. $\text{pneumoniae}$ in suspension formed O₂^? in the presence of NADH and flavins and extracts of $\text{M}$. $\text{pneumoniae}$ formed O₂^? in the presence of either NADH or NADPH. The lack of superoxide dismutase in $\text{M}$. $\text{pneumoniae}$ could not be attributed to superoxide dismutase in the complex medium in which the organisms were grown because organisms grown in medium in which the superoxide dismutase had been inactivated by heat still contained undetectable amounts. Mycoplasmas appear to be an exception to the rule that organisms which consume O₂ synthesize superoxide dismutase.     

Sizing of two bacteriophage T4 specific messenger ribonucleic acids formed in vitro and in vivo

Messenger RNA for two T4 specific enzymes, deoxynucleotide kinase and α glucosyltransferase, have been sized by sedimentation on sucrose density gradients. The sedimentation constants of transferase and kinase mRNAs formed $\text{in vitro}$ were 21.5S and 14.5S respectively, regardless of the duration of incubation up to 20 min. Although the kinase mRNA isolated from cells infected with T4 phage for 10 min was the same size as found $\text{in vitro}$ (14.5S), the transferase mRNA was found in a segment approximating the size of the kinase mRNA (14.5S). The experiments indicate that α glucosyltransferase mRNA is formed first as a polycistronic message and is then processed to the smaller unit.     

Evidence for the existence of a ubiquinone protein and its radical in the cytochromes b and c1 region in the mitochondrial electron transport chain.

A highly purified cytochrome $\text{b}\text{?}\text{c}{}_1$ complex which is free of QP_s (see BBRC $\text{78}\text{, 259 and}\text{79}$, 939) yields 20–30% of semiubiquinone (based on the total Q content in the system) in the presence of catalytic amounts of QP_s and succinate dehydrogenase (at or lower than 2 × 10^{?9 M}. The radical shows typical $\text{g}\text{= 2.00}$ signals with line widths of 8 and 9 gauss, respectively, at about 22°C and 77°K. The appearance of the radicals approximately parallels that of $\text{b}$ reduction but not its disappearance. However, addition of theonytrifluoroacetone or antimycin A immediately abolishes both radical formation and $\text{b}$ reduction. These and other observations indicate that the true carrier property of Q is through its binding with proper proteins but not the protei-free form.     

Detection of a phase transition in red cell membranes using positronium as a probe

Positron lifetimes in human red cell ghost membranes have been measured as a function of temperature from 3°C to 25°C. A marked sudden change in the $\text{ortho-}\text{positronium}$ annihilation rate was found at 16–18°C during the heating cycle and at 18–20°C in the cooling cycle. Such sudden change of microenvironment in the membranes sensed by $\text{ortho-}\text{positronium}$ is attributed to the sudden change of water diffusion rate through the membranes which is a consequence of the sudden change in free volume, or fluidities in the lipid layers.