Veal heart ribonuclease P has an essential RNA component

The activity of RNase P (EC 3.1.26.5) from veal heart can be abolished by pretreatment of the enzyme preparation with micrococcal nuclease, pancreatic RNase A, or RNase T₁. This indicates that veal heart RNase P contains an RNA component essential for function of the enzyme as has also been shown for $\text{E. coli}$ RNase P (1–3). Additionally, veal heart RNase P has a buoyant density in Cs₂SO₄ of 1.33 g/cm³, which is intermediate between that of protein and nucleic acid.     

Ribonuclease F,a putative processing endoribonuclease from Escherichia coli

A new endoribonuclease activity, RNase F, was partially purified from $\text{Escherichia coli}$ cells. This activity can cleave a precursor RNA molecule (of Species 1), isolated from T4 infected cells, in a specific site. This activity is different from the other three know processing endoribonucleases of $\text{E. coli}$ RNase III, RNase E and RNase P.     

5' Cap methylation of homologous poly A(+) RNA by a RNA (guanine-7) methyltransferase from Neurospora crassa.

RNA (guanine-7) methyltransferase, partially purified from $\text{N.}\text{crassa}$ mycelia, catalyzed the transfer of the methyl group from S-adenosylmethionine to the 5′ terminus of both $\text{N.}\text{crassa}$ poly A(+) RNA and reovirus unmethylated mRNA. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated poly A(+) RNA from $\text{N.}\text{crassa}$ yielded the “cap” structures m ⁷G(5′)pppAp and m ⁷G(5′)pppGp in a ratio of 2:1 respectively. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated reovirus mRNA yielded m ⁷G(5′)pppGp exclusively. The absence of mRNA 2′-0-methyltransferase activity in the enzyme preparation is consistent with the absence of 2′-0-methylation in $\text{N.}\text{crassa}$ mRNA [Seidel, B. L. and Somberg, E. W. (1978) Arch. Biochem. Biophys. $\text{187}$, 108–112]. This is the first isolation of an eucaryotic, cellular RNA (guanine-7) methyltransferase that has been shown to methylate homologous substrate.     

Ultraviolet light-induced recombination

Stimulation of transduction in $\text{Escherichia coli}$ by ultraviolet irradiation of the transducing phage P1 requires the $\text{uvrA-uvrB}$ nuclease but not the $\text{uvrC}$ product or DNA polymerase I. It is hypothesized that the first step in “normal” recombination can be bypassed by any procedure generating single-stranded ends of DNA (as, for example, by $\text{uvra-uvrB}$ nuclease activity).     

Mechanism of interferon action partial purification and characterization of a low-molecular-weight interferon-mediated inhibitor of translation with nucleolytic activity

A low-molecular-weight interferon-mediated ribosome-associated inhibitor of reovirus mRNA translation was purified from the 0.5 $\text{M}$ KCl ribosomal salt-wash fraction of mouse L₉₂₉ cells. The inhibitor possessed nucleolytic activity with reovirus [³H]mRNA as a substrate. Loss of translational inhibitory activity correlated with the thermal inactivation of the nuclease. A low-molecular-weight ($\text{<}$10K) component present in the Bio-Gel P150 chromatography fractions which contained the interferon-mediated nucleolytic activity was labeled in vivo with [¹⁴C]valine; a smaller component present in the same fractions was phosphorylated in vitro with [γ-³²P]ATP. The $\text{<}$10K components were resolved from ～50K, ～30K and ～20K phosphorylatable proteins associated with ribosomes that possess the interferon-mediated inhibitor(s) of viral mRNA translation.     

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.     

A protein-bound form of thioacetamide in liver nucleoli

The cellular distribution of ³⁵S from ³⁵S- thioacetamide was determined in rabbit liver subcellular fractions following its $\text{in vivo}$ administration. Of the various fractions isolated, only the nucleolar fraction contained ³⁵S counts that were insoluble in 10% trichloroacetic acid but soluble in trichloroacetic acid if the fraction was treated with trypsin but not RNase or DNase. These results demonstrate that a protein bound form of thioacetamide is present in the nucleolus following $\text{in vivo}$ administration of this drug.     

Pyridoxal 5' phosphate: a selective inhibitor of oncornaviral DNA polymerases.

Pyridoxal 5′ phosphate at concentrations < 0.5 mM inhibits $\text{polymerization}$ of deoxynucleoside triphosphate catalysed by variety of DNA polymerases isolated from type C RNA tumor viruses, as well as $\text{E.}\text{coli}$, but $\text{does}\text{not}$ affect the polymerase associated RNase H activity. Both phosphate and aldehyde groups of pyridoxal phosphate are essential for the inhibition which appears to be mediated through the reversible Schiff base.     

Induction of single-strand regions in nuclear DNA by adriamycin.

Incubation of adriamycin with isolated nuclei converts nuclear DNA to a form which is susceptible to hydrolysis by $\text{Neurospora}$$\text{crassa}$ nuclease an enzyme highly specific for the cleavage of single-stranded DNA. The effect of adriamycin on nuclear DNA incubated in the presence of the nuclease can be determined by measuring the release of acid-soluble nucleotides or by analyzing the DNA after centrifugation in neutral sucrose gradients. Similar changes in chromatin structure are not observed during incubation of nuclei with adriamycin alone. In addition to adriamycin, daunomycin and ethidium bromide are also active in inducing the formation of DNA structures which are susceptible to the $\text{Neurospora}$$\text{crassa}$ nuclease. The results suggest that certain antitumor agents can induce the formation of single-strand regions in nuclear DNA and that these sites probably occur as a result of a DNA strand separating event.     

Base-group specificity at the primary recognition site of ribonuclease T1 for minimal RNA substrates

Kinetic studies on the RNase T₁-catalyzed transesterification of 12 dinucleoside monophosphates, N_p¹N² (N¹ = A, C, and U; N² = A, C, G, and U) at pH 5, 25 °C, and 0.2 m ionic strength, revealed that the catalytic efficiency ($\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$) for GpN substrates (H. L. Osterman, and F. G. Walz, Jr., 1978, Biochemistry, 17, 4142) was ~10⁶-fold greater than corresponding ApNs and at least 10⁸-fold greater than corresponding CpNs and UpNs. The catalytic activity with ApN substrates survives phenol extraction which indicates (along with other criteria) that it is intrinsic to RNase T₁ and is not due to trace contamination by other nucleases. Circumstantial evidence is presented which suggests that homologous GpN and ApN substrates bind productively at different sites on the enzyme. The results of steady-state kinetic studies of RNase T₁ with IpNs (N = C and U) were compared with those for GpNs and indicated that the primary effect of the guanine 2-NH₂ group is to enhance substrate binding at the primary recognition site by ~2.6 kcal/mol. Values of ($\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$) showed the order NpC > NpU (N = A, G, and I) which evidences the existence of a subsite for the leaving nucleoside group that prefers cytidine: interactions at this subsite are reflected in k_cat rather than K_m.     

Detection of a DNA polymerase β stimulating protein in hela cell nuclear extracts

HeLa cell nuclei contain a protein which stimules the $\text{in}\text{vitro}$ activity of HeLa cell DNA polymerase β, but does not affect the activity of DNA polymerase α and γ. The protein, which binds to both single- and double-stranded DNA, does not possess nuclease activity and is heat stable, surviving 100 degrees C for 10 min. The molecular weight of the protein is approximately 85,000 and evidence is presented that it may exert its stimulatory effect by direct interaction with β-polymerase.     

Evidence for a nuclease in Saccharomyces cerevisiae that affects the cell-free translation of natural messenger RNA.

A postpolysomal extract of $\text{Saccharomyces}\text{cerevisiae}$, treated with micrococcal nuclease to remove endogenous mRNAs, translates exogenous natural and synthetic mRNA templates actively and accurately at 20°C. When the temperature of incubation is 30°C or higher, protein synthesis with yeast poly(A)⁺ mRNA is markedly reduced, but synthesis of polyphenyl-alanine with poly (U) is only slightly affected. The protein synthesizing activity of the extract is decreased 50% in 30 minutes at 37°C, while the ability of yeast mRNA to template for protein synthesis is decreased 50% in 5 to 7 minutes when it is incubated with the postpolysomal fraction at 37°C. The release of radioactivity from isotopically-labeled yeast mRNA, into the acid-soluble form, is also much greater at 37°C than at 20°C. Thus, at the elevated temperatures, the loss of mRNA templating activity and RNA hydrolysis occur more rapidly than the loss of activity of the translational apparatus. The evidence suggests that the failure of the extract to catalyze translation at 30°C or higher, as compared to 20°C, is due to a temperature-stimulated nuclease that degrades mRNA.     

The role of the sites for ATP of the Ca2+-ATPase from human red cell membranes during Ca2+-phosphatase activity

1. In the presence of ATP, the Ca²⁺ pump of human red cell membranes catalyzes the hydrolysis of $\text{p-}\text{nitrophenyl}$ phosphate. The requirement for ATP of the Ca²⁺-$\text{p-}\text{nitrophenylphosphatase}$ activity was studied in relation to the two classes of site for ATP that are apparent during Ca²⁺ -ATPase activity. 2. (a) The $\text{K}{}_{0.5}$ for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ extrapolated at 0 mM PNPP is equal to the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the Ca²⁺ -ATPase. (b) PNPP competes with ATP and its effectiveness is the same regardless the nucleotide acts as the substrate of the Ca²⁺ -ATPase or as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 3. PNPP at the high-affinity site does not substitute for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 4. At ATP concentrations that almost saturate the high-affinity site, Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity increases as a function of PNPP along an S-shaped curve, while Ca²⁺ -ATPase activity is partially inhibited along a curve of the same shape and apparent affinity. The fraction of Ca²⁺ -ATPase activity which is inhibited by PNPP is that which results from occupation of the low-affinity site by ATP. 5. Activation of the Ca²⁺ -ATPase by ATP at the low-affinity site is associated with inhibition of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity. Both phenomena take place with the same apparent affinity and along curves of the same shape. 6. Experimental results suggest that: (a) the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity depends on ATP at the high-affinity site; (b) PNPP is hydrolyzed at the low-affinity site; (c) Ca²⁺ -ATPase activity at the high-affinity size persists during Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity.     

Conformational states of enkephalins in solution.

Chicken erythrocyte chromatin was partially digested with micrococcal nuclease and separated into multimeric subunit fractions by gel permeation chromatography. The fractions were characterized by their Svedberg constant, diffusion coefficient, circular dichroism, and electrophoresis pattern of the extracted DNA. The molecular weight dependence of the sedimentation coefficient was found to be S_20,w = .011 × M^.554. The molecular weight dependence of $\text{rmf}\text{f}{}_{\mathrm{o}}$ is best represented in the Kirkwood theory by either a helical superstructure $\text{or}$ a flexible coil $\text{with}\text{attractive}\text{interactions}$ between nucleosome units. The dimer calculations of $\text{f}\text{f}{}_{\mathrm{o}}$ suggest that the core particles are separated by spacer regions which contribute up to ～20% of the frictional properties of the molecule.     

Cytoplasmic poly(A) polymerase from sea urchin eggs, merogons, and embryos

The presence of cytoplasmic poly(A) polymerase has been established in sea urchin eggs and four-cell embryos by subcellular fractionation and use of enucleate egg halves. ATP is the only ribonucleoside triphosphate incorporated. This incorporation is time dependent, contingent on input protein concentration, and immune to a variety of antimetabolites known to inhibit DNA-directed RNA synthesis. Both the unfertilized egg and the four-cell embryo cytoplasmic poly(A) polymerase activities display a preference for Mn²⁺. While oligo(A)₄ is inactive as a primer, addition of $\text{oligo(A)}{}_{\mathrm{<mtext>16</mtext>}}\text{,}\text{poly(A)}{}_{\mathrm{<mtext>45</mtext>}}$ and $\text{poly(A)}{}_{\mathrm{<mtext>90</mtext>}}$ stimulates ATP incorporation. On a unit per milligram protein basis, the endogenous activity associated with cytoplasmic fractions obtained from nucleate and enucleate egg halves is 36 and 83% that obtained with the cytoplasmic fraction prepared from the unfertilized egg. In the presence of $\text{oligo(A)}{}_{\mathrm{<mtext>16</mtext>}}$, both the nucleate and enucleate egg halves exhibit 81% of the activity associated with the unfertilized egg cytoplasmic fraction. The level of Mn²⁺ cytoplasmic poly(A) polymerase activity from the four-cell embryo is approximately 50% that of the unfertilized egg. This decrease does not appear to be due to either a postfertilization alteration in the subcellular localization of poly(A) polymerase or an increase in RNase activity. Supplementation with $\text{oligo(A)}{}_{\mathrm{<mtext>16</mtext>}}$ failed to restore the four-cell embryo cytoplasmic poly(A) polymerase potential to a level comparable to that of the unfertilized egg. Suppression of postfertilization protein synthesis by emetine, however, prevents this developmental decline in ATP incorporation thereby suggesting that postfertilization cytoplasmic poly(A) polymerase activity is subject to negative translational control.     

Demethylation of O6-methylguanine in a synthetic DNA polymer by an inducible activity in Escherichiacoli

$\text{O}$⁶-Methyl[8-³H]deoxyguanosine in a synthetic DNA polymer, poly(dC, dG, m⁶dG), is demethylated by cell-free extracts of $\text{Escherichia}\text{coli}\text{B}\text{r}$ adapted by exposure to $\text{N}$-methyl-$\text{N}$′-nitro-$\text{N}$-nitrosoguanidine, as shown by the appearance of ³H-labeled deoxyguanosine in hydrolysates of the recovered DNA. The demethylating activity could not be detected in extracts of nonadapted $\text{E}\text{.}\text{coli}$. These results provide direct evidence that a previously described inducible repair activity in $\text{E}\text{.}\text{coli}$ acts by demethylating $\text{O}$⁶-methylguanine at the DNA level.     

Synthesis and biological activity of glycosylated analogs of somatostatin

The synthesis by solid-phase methodology of two glycosylated analogs of somatostatin [Glc-Asn⁵]-SS and [NAcGlc-Asn⁵]-SS is described. These two analogs have been biologically tested on the secretion of pituitary growth hormone, pancreatic glucagon and insulin. The results show that glycosylation of somatostatin on the Asn⁵ residue decreases by a hundred fold the inhibition activity on GH release when tested $\text{in}$$\text{vitro}$. $\text{In}$$\text{vivo}$, since the activity is similar to somatostatin the carbohydrates are probably removed by some enzymatic reaction and thus liberate the full activity of somatostatin.     

Structural alteration of rRNA in the L7/L12 region of 50s ribosome on removal of L7/L12 proteins

Core particles of 50S ribosomes depleted of $\text{L7}\text{L12}$ proteins are degraded by RNase I at a considerably slower rate than intact 50S ribosomes. The normal rate is restored on incorporating $\text{L7}\text{L12}$ proteins into the core particles. The capacity of the core particles to inhibit the RNase I-catalyzed hydrolysis of poly A and to bind ethidium bromide is also greater with core particles than with intact 50S ribosomes. It appears from these results that the region(s) of rRNA in the vicinity of $\text{L7}\text{L12}$ proteins has less ordered structure which, on removal of $\text{L7}\text{L12}$ proteins, becomes more organized. Apparently, binding of $\text{L7}\text{L12}$ proteins to the 50S core leads to the destabilization of double-stranded regions of rRNA.     

Modified polynucleotides. V. Slow-down of nuclease action by 5-alkyluracil-containing DNAs

Poly d/[³H]A-r⁵U/ type of synthetic models of bacteriophage DNAs containing thymine analogues were prepared by DNA polymerase and tested for stability against nucleases /$\text{r}$ was a n-alkyl group from methyl to pentyl/. The 5-pentyluracil-containing copolymer was found to be most stable: 50 % degradation with pancreatic DNase, spleen DNase, snake venom phosphodiesterase or micrococcal nuclease required 3–15 times as much time as that of poly d/A-T/.