The primer specificity of cytoplasmic poly(A) polymerase from cryptobiotic gastrulae of Artemia salina

Poly(A) polymerase has been purified to near homogeneity from the cytoplasm of Artemia salina as described previously (Roggen, E and Slegers, H. (1985) Eur. J. Biochem. 147, 225–232). Affinity chromatography on poly(A)-Sepharose 4B separates the enzyme preparation into two fractions. In standard assay conditions poly(A) polymerase fraction I (poly(A)-Sepharose 4B unbound) and fraction II (poly(A)-Sepharose 4B bound) have specific activities of 2.4 and 8.0 μmol AMP/h per mg enzyme, respectively. Poly(A) polymerase fraction II shows a high primer specificity towards the 17 S poly(A)-containing mRNP. Depending on the reaction conditions used, poly(A) sequences of 140 ± 15 AMP residues/μg enzyme are synthesized on the latter primer. In contrast, poly(A) polymerase fraction I only elongates oligo(A) primers efficiently. An endogenous RNA is detected in poly(A) polymerase II preparations. This RNA has a length of 83 ± 2 nucleotides and is a component of a 60 kDa particle. After removal of the latter the specificity of poly(A) polymerase fraction II for the 17 S poly(A)-containing mRNP is abolished and the characteristics of the enzyme resemble those of poly(A) polymerase I.     

Two distinct poly(A) polymerases isolated from the cytoplasm of Ehrlich ascites tumour cells

The poly(A) polymerases from the cytosol and ribosomal fractions of Ehrlich ascites tumour cells are isolated and partially purified by DEAE-cellulose and phosphocellulose column chromatography. Two distinct enzymes are identified: (a) a cytosol Mn2+-dependent poly(A) polymerase (ATP:RNA adenylyltransferase) and (b) a ribosome-associated enzyme defined tentatively as ATP(UTP): RNA nucleotidyltransferase. The cytosol poly(A) polymerase is strictly Mn2+-dependent (optimum at 1 mM Mn2+) and uses only ATP as substrate, poly(A) is a better primer than ribosomal RNA. The purified enzyme is free of poly(A) hydrolase activity, but degradation of [3H]poly(A) takes place in the presence of inorganic pyrophosphate. Most likely this enzyme is of nuclear origin. The ribosomal enzyme is associated with the ribosomes but it is found also in free state in the cytosol. The purified enzyme uses both ATP and UTP as substrates. The substrate specificity varies depending on ionic conditions: the optimal enzyme activity with ATP as substrate is at 1 mM Mn2+, while that with UTP as substrate is at 10--20 mM Mg2+. The enzymes uses both ribosomal RNA and poly(A) [but not poly(U)] as primers. The purified enzyme is free of poly(A) hydrolase activity.     

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.     

Subcellular distribution and properties of poly(A)-containing RNA from cultured plant cells.

Cultured sycamore cells rapidly incorporate [3H]uridine or [32P]orthophosphate into rRNA precursors and polydisperse RNA. Mature rRNA accumulates only after a lag period of approximately 40 min. Fractionation of pulse-labelled cells and analysis of the RNA shows that after 30 min the rRNA precursors, together with some polydisperse RNA, are confined to the nucleus. In consequence radioactive polydisperse RNA can be isolated from polyribosomes in the complete absence of labelled rRNA. Approximately 40% of this RNA is retained by an oligo(dT)-cellulose column and by this criterion is judged to contain poly(A) sequences. A smaller proportion of nuclear polydisperse RNA also contains poly(A). The tendency for poly(A)-containing RNA to aggregate complicates molecular weight determinations. Denaturation of poly(A)-containing RNA in 8 M urea prior to gel electrophoresis produces a broad peak of RNA with an average Mr = 10(6). Analysis of the nucleotide composition of total cell poly(A)-containing RNA shows that it contains 41% AMP. Roughly 6% of this RNA is resistant to digestion by ribonuclease A and T1. AMP is the only nucleotide detectable in these fragments. From their mobility during electrophoresis in 8 M urea at 60 degrees C with 5.8-S, 5-S and tRNA as molecular weight markers it is concluded that the poly(A) regions contain an average of 160 nucleotides.     

Turnover of polyadenylate-containing ribonucleic acid in Saccharomyces cerevisiae.

We examined the kinetics of incorporation of [3H]adenine into polyadenylate-containing ribonucleic acid [poly(A)-containing RNA] in yeast. The total poly(A)-containing RNA from spheroplasts and intact cells and the polysomal poly(A)-containing RNA exhibited similar incorporation kinetics. At 30 C half-saturation of the pool of poly(A)-containing RNA with label occurred in approximately 22 min. Since precursor pools appeared to require 5 min to saturate with label, we conclude that at 30 C messenger RNA molecules in yeast decay with an average half-life of 17 min.     

Comparison of poly(A)-containing RNAs in different cell types of the lower eukaryote Schizophyllum commune

Poly(A)-containing RNAs were isolated from morphologically different cells of the fungus Schizophyllum commune. Using mRNA markers the number-average length of poly(A)-containing RNA in total RNA and in purified poly(A)-containing RNA was estimated as 1100 nucleotides. Number-average length of poly(A)-tracts was 33 nucleotides. 2.5% of total RNA is poly(A)-containing RNA and probably up to 7.5% are non-polyadenylated polydisperse RNA sequences. Saturation hybridization of poly(A)-containing RNA to gap-translated [3H]DNA resulted in 16% of the reactive single-copy DNA to become S1 nuclease resistant. It was found that purified poly(A)-containing RNA represented the entire RNA complexity, i.e. 10 000 different RNA sequences in S. commune. RNA sequences isolated from morphologically different mycelia and from fruiting and non-fruiting mycelia were identical for at least 90%.     

Extensive homology between poly(A)-containing mRNA and purified nominal poly(A)-lacking mRNA in mouse kidney

To investigate poly(A)-lacking mRNA in mouse kidney, we studied a fraction of renal mRNA that does not bind to oligo(dT)-cellulose but can be purified by benzoylated cellulose chromatography. Nominal poly(A)-lacking mRNA and poly(A)-containing mRNA have complete nucleotide sequence homology, suggesting that kidney does not contain mRNAs that are not represented in the polyadenylated RNA fraction. Translation products directed by nominal poly(A)-lacking mRNA and poly(A)-containing mRNA are qualitatively and quantitatively similar in one-dimensional polyacrylamide gels. [3H]cDNA transcribed from poly(A)-containing mRNA hybridizes with its template and with nominal poly(A)-lacking mRNA to the same extent (95%) and with the same kinetics; reaction of [3H]cDNA to nominal poly(A)-lacking mRNA with the two mRNA populations gives the same result. The extensive homology these two mRNA populations share is important to the interpretation of mRNA lifetime and to the analysis of authentic poly(A)-lacking mRNAs.     

The binding of Met-tRNAf to isolated 40-S ribosomal subunits and the formation of Met-tRNAf - 80-S-ribosome initiation complexes.

Different forms of 40-S ribosomal subunit, distinguishable by their buoyant densities on CsCl equilibrium density gradients, are formed when derived 40-S ribosomal subunits are incubated with partially purified reticulocyte ribosomal wash proteins. One of these subunits, the 1.37-g-cm-3 form is not present in the cell but the other two forms, the 1.40-g-cm-3 and 1.40-g-cm-3 subunits, are present in cell extracts. 35S label is bound to 1.37-g-cm-3 and 1.40-g-cm-s subunits when [35S]Met-tRANf, GTP and poly(A,U,G) are included in the incubations. The 35S-labelled 40-S subunits recovered, and the amount of 35S label bound to them, are changed if the [35S]Met-tRNAf-40-S-subunit-poly(A,U,G) complexes are first purified on sucrose gradients before analysing them on CsCl. The 1.37-g-cm-3 particle is no longer seen and the total quantity of 35S label on the 40-S subunits is 90% lower after sucrose gradient purification. Between 30% and 40% of the 40-S subunits bind [35S]Met-tRNAf when 1 mM GTP, an excess of ribosomal wash proteins and [35S]Met-tRNAf over derived 40-S subunits, and poly(A,U,G) or AUG is included in the incubations. The omission of poly(A,U,G) or AUG from the incubations substantially lowers the amount of subunit-bound 35S label ultimately recovered. With these incubations less than 10% of the 40-S subunits have bound [35S]Met-tRNAf. [35S]Met-tRNAf binding is affected by the nature of the RNA added. The addition of poly(U), rRNA and native 9-S golbin mRNA is without effect, whereas denatured globin mRNA is stimulatory. Maximum binding is obtained however with AUG. Poly(A,U,G) is less stimulatory than AUG but more stimulatory than denatured mRNA, suggesting that the number as well the accessibility of the AUG initiations condons determines the amount of 35S label bound. Similar results are obtained for the ribosomal-wash-dependent binding of [35S]Met-tRNAf to 80-S ribosomes. Contrary to the binding results, the ability of mRNA to stimulate protein synthesis is dependent on the integrity of the mRNA. Thus, native 9-S globin mRNA but not poly(A,U,G) stimulatex protein synthesis in the wheat germ system. HCHO-treated globin mRNA, although stimulatory, is 45% less effective than native mRNA. The addition of AUG, derived 60-S subunits and extra ribosomal wash is required for the formation of [35S]Met-tRNAf-80-S-ribosome complexes from sucrose-gradient-purified [35S]Met-tRNAf-40-S-subunit complexes. The 80-S ribosome complexes are able to form peptide bonds. Thus, if puromycin is added to the full incubations at zero time, no 35S label is present on the 80-S ribosome. 35S label is released as methionyl-puromycin. If the [35S]Met-tRNAf-40-S-subunit complexes are assembled with poly(A,U,G) or AUG in the incubations and then purified, only derived 60-S subunits are required to form [35S]Met-tRNAf-80-S-ribosome complexes. 35S label is not released from them when puromycin is added to the incubations unless extra ribosomal wash is also added.     

Regulation of poly(A) polymerase activity and poly(A)+ RNA in germinated wheat embryos under conditions of pathogenesis

The induction of poly(A) polymerase was accompanied by a rise in the level of poly(A)⁺ RNA during early germination of excised wheat embryos (48 h). Fractionation of this RNA-processing enzyme by acrylamide gel electrophoresis and also by molecular sieving on Sephadex G-200 revealed a single molecular form of poly(A) polymerase with a molecular weight of 125 000. Wheat poly(A) polymerase specifically catalyzed the incorporation of [³H]AMP from [³H]ATP into the polyadenylate product only in the presence of primer RNA. Substitution of [³H]ATP by other labelled nucleoside triphosphates, such as [³H]GTP, [³H]UTP or [α-³²P]CTP in the assay mixture did not yield any labelled polynucleotide reaction product. The ³H-labelled reaction product was retained on poly(U)-cellulose affinity column and was not degraded by RNAase A and RNAase T₁ treatment. In addition, the nearest-neighbour frequency analysis of the ³²P-labelled reaction product predominantly yielded [³²P]AMP. Thus, characterization of the reaction product clearly indicated its polyadenylate nature. The average chain length of the [³H]poly(A) product was 26 nucleotides. Infection of germinating wheat embryos by a fungal pathogen (Drechslera sorokiana) brought about a severe inhibition (62–79%) of poly(A) polymerase activity. Concurrently, there was a parallel decrease (73%) in the level of poly(A)⁺ RNA. Inhibition of poly(A) polymerase activity in infected embryos could be due to enzyme inactivation, which in turn brought about a downward shift in the level of poly(A)⁺ RNA. The crude extract of the cultured pathogen contains a non-dialysable, heat-labile factor, which, along with a ligand, inactivates (65–74%) poly(A) polymerase in vitro. The fungal extracts also contained a dialysable, heat-stable stimulatory effector which activated wheat poly(A) polymerase (3.6–4.0-fold stimulation) in vitro. However, the stimulatory fungal effector was not expressed in vivo, but was detectable after the inhibitory fungal factor had been destroyed by heat-treatment in our in vitro experiments.     

Different binding of poly(A)-containing and poly(A)-free fractions of nuclear ribonucleic acid to ribosomes from rat liver.

Total nuclear RNA extracted from nuclei of rat liver cells by phenol/chloroform in the presence of sodium dodecyl sulphate was separated by combined gel filtration on Sepharose 4 B and affinity chromatography on poly(U) Sepharose into fractions differing in their molecular weights and contents of poly(A) sequences. The poly(A)-containing 45-S RNA became labelled most rapidly if rats were administered [3H] orotic acid. This fraction showed a high template activity when added to postmitochondrial supernatants of the Krebs ascites tumour. Fractions of nRNA, free of poly(A) sequences, had no stimulating effect on protein synthesis in this system. The 45-S RNA-containing poly(A) was readily bound to crude polyribosomes from rat liver at 0 degrees C and both ATP and GTP were necessary for this reaction. Sucrose gradient analyses provided evidence that this RNA species is bound predominantly to 80-S ribosomes. No binding was obtained with polyribosomes washed with 0.5 M KCl. The binding ability of washed polyribosomes was restored by the addition of the ribosomal wash fraction or rat liver cytosol. Crude polyribosomes bound significantly lower quantities of nRNA species free of poly(A) when compared with poly(A)-45-S RNA. The label was scattered through the whole ribosomal sedimentation pattern with no predominant peaks and the binding reaction required neither soluble factors nor nucleotide cofactors. The labelling kinetics and high template activity of poly(A)-45-S nRNA indicate that this fraction contains precursors of cytoplasmic mRNA. Requirements for soluble factors and nucleotide cofactors in the binding of this RNA species to 80-S ribosomes suggest that this binding, unlike that of other nRNA species, has a specific mechanism resembling that of mRNA binding during peptide initiation.     

Poly(A) polymerase in quail oviduct. Changes during estrogen induction.

A nuclear poly(A) polymerase has been isolated from oviducts of immature quails. It could be purified 4300-fold. The enzyme depends specifically on ATP as substrate and requires Mg2+. The most effective primer for the enzyme is a polynucleotide, isolated from oviduct tissue. A poly(A) sequence to a maximum of 60 AMP residues is covalently linked per primer molecule. The poly(A)-rich product of the enzymatic reaction can be annealed to oligo(dT)-cellulose. The purest fraction does not contain any detectable poly(A)-degrading enzyme activity. Only very low activities of RNA polymerase are present. The poly(A polymerase activity in the assay with ATP is reduced by the ATP analogue, beta, lambda-ATP-methylene-diphosphonate. Both K-m and V are lowered. The ATP analogue is incorporated to a smaller extent into the poly(A) sequence, synthesized by the enzyme. Several other analogues of adenine, adenine nucleosides and adenine nucleotides are without effect on the enzymatic reaction. By these properties poly(A) polymerase can be distinguished from RNA polymerases form I and form II, isolated from the same tissue. Actinomycin D and alpha-amanitin failed to inhibit poly(A) polymerase activity. The activity of poly(A) polymerase has been determined during primary stimulation with the estrogen analogue diethylstilbestrol (daily injection for 5 days), after withdrawal of the hormone for 17 days and after secondary stimulation with the hormone analogue. The enzyme activity does not change during primary stimulation, withdrawal of the hormone or secondary stimulation. However the activity of a poly(A) degrading enzyme, localized in the nucleus, is reduced in oviducts from hormone-treated quails.     

Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis

A kinetic analysis of the appearance of [3H]uridine label in RNA sequences that neighbor poly(A), as well as the incorporation of [3H]adenosine label into both the RNA chain and the poly(A) of poly(A)-containing molecules, shows that poly(A) is added within a minute or so after RNA chain synthesis in Chinese hamster ovary cells and HeLa cells. Previous conclusions by several groups (5-7) that poly(A) might be added as long as 20-30 min after RNA synthesis appear to be in error, and the present conclusion seems much more in line with several different types of recent studies with specific mRNAs that suggest prompt poly(A) addition (13-16).     

Methylated messenger RNA in mouse kidney.

Polyadenylated messenger RNA from mouse kidney labeled in vivo exhibited a pattern of methylation distinct from that of rRNA and tRNA. After mice were given L-[methyl-3H]methionine, 4% of the polyribosomal RNA label was bound to oligo (dT)-cellulose; 20-24% of orotate- or adenine-labeled polyribosomal RNA eluted in the poly(A)+ RNA fraction under similar conditions. [3H]Methyl radioactivity was not incorporated into low molecular weight (5-5.8 S) rRNA, indicating the extent of nonmethylpurine ring labeling was negligible. [3H]Methyl-labeled poly(A)+ RNA sedimented heterogeneously in sodium dodecyl sulfate containing gradients similarly to poly(A)+ mRNA labeled with [3H]orotic acid. Based on an average molecular length of 2970 nucleotides, renal mRNA was estimated to contain 8.6 methyl moieties per molecule. Analysis of alkaline-hydrolyzed RNA sampled by DEAE-Sephadex-urea chromatography provided estimates of the relative amounts of base and ribose methylation. Although 83% of the [3H]methyl radioactivity in rRNA was in the 2'-0-methylnucleotide fraction, no methylated dinucleotides were found in mRNA. In poly(A)+ mRNA 60% of the [3H]methyl label was in the mononucleotide fraction; the remainder eluted between the trinucleotide and tetranucleotide markers and had a net negative charge between -4 and -5. The larger structure, not yet charcterized, could result from two or three consecutive 2'-0-ribose methylations and is estimated to contain 2.6 methyl residues. Alternatively, the oligonucleotide could be a 5'-terminal methylated nucleotide species containing 5'-phosphate(s) in addition to the 3'-phosphate moiety resulting from alkaline hydrolysis. Either structure could have a role in the processing or translation of mRNA in mammalian cells.     

Cell-free synthesis of tumor-type poly(A) polymerase

Previous studies in this laboratory suggested that in adult liver, either the gene for the tumor-type poly(A) polymerase is poorly transcribed or the mRNA for this enzyme is largely not expressed. To test these possibilities, total RNA from rat liver and Morris hepatoma 3924A RNA were isolated by using a guanidine thiocyanate method; poly(A+) RNA and poly(A-) RNA were separated by oligo(dT)-cellulose chromatography and used for translation in a rabbit reticulocyte lysate system. After in vitro translation, the products were immunoprecipitated with either purified anti-tumor poly(A) polymerase antibodies or control immunoglobulins. When the polypeptides translated from poly(A+) or poly(A-) hepatoma RNA were precipitated with immune sera, a unique [35S]methionine-labeled 35-kilodalton (kDa) protein was observed. This band was not apparent when control serum was used for the immunoprecipitation. The radiolabeled 35-kDa polypeptide was not evident when the products were incubated with highly purified tumor nuclear poly(A) polymerase prior to immunoprecipitation. Prior incubation of the translation products with bovine serum albumin instead of poly(A) polymerase had no effect on the immunoprecipitation. This 35-kDa protein was not apparent when liver poly(A+) RNA was used to direct translation. These data demonstrate that (a) the tumor enzyme is not synthesized as a precursor, (b) tumor mRNA, but not normal liver mRNA, contains detectable sequences coding for tumor-type poly(A) polymerase, and (c) poly(A) polymerase mRNA also exists as a poly(A-) population.     

Isolation and characterization of poly(adenylic acid)-containing messenger ribonucleic acid from rat liver polysomes

Undegraded rat liver polysomes were obtained after homogenizing the tissue in a medium containing NH4Cl, heparine, and yeast tRNA. Purification of poly(A)-containing RNA from polysomal RNA was accomplished by affinity chromatography on oligo(dT)-cellulose columns. Poly(A)-containing RNA molecules were monitored by the formation of ribonuclease-resistant hybrids with [3H]poly(U). To improve the separation of messenger RNA and ribosomal RNA by oligo(dT)-cellulose it was found essential to dissociate the aggregates formed between both molecular species by heat treatment in the presence of dimethylsulfoxide (Me2SO) prior to chromatography. Sucrose gradient analysis under denaturing conditions showed that the preparations obtained were virtually free of ribosomal RNA. Poly(A)-containing RNA constituted approx. 2.2% of the total polysomal RNA and the number average size was 1500--1800 nucleotides, as judged by sedimentation analysis on sucrose density gradients containing Me2SO. Approximately 8.2% of the purified preparation obtained was able to anneal with [3H]poly(U); the number average nucleotide length of the poly(A) segment of the RNA population was calculated to be 133 adenylate residues. Based on these values, our preparations appear to be greater than 90% pure. The RNA fractions obtained after oligo(dT)-cellulose chromatography were used to direct the synthesis of liver polypeptides in a heterologous cell-free system derived from wheat-germ. The system was optimized with respect to monovalent and divalent cations, and presence of polyamines (spermine). More than 65% of the translational activity present in the unfractionated polysomal RNA was recovered in the final poly(A)-containing RNA fraction. However, about 25% of the activity was found to be associated with the unbound fraction which was essentially free of poly(A)-containing RNA. Immunoprecipitation analysis with a specific antiserum to rat serum albumin demonstrated that about 6--8% of the labeled synthetic products translated from the poly(A)-containing RNA sample corresponded to serum albumin. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a heterogeneous distribution of molecular sizes ranging from 15 000 to greater than 70 000 daltons. Spermine not only increased the overall yield and extent of protein synthesis, but also resulted in higher yields of large protein products. Under optimal translation conditions a discrete peak representing about 7% of the total radioactivity was observed to migrate with rat serum albumin.     

Adenosine diphosphate ribosylation of histone H1 by purified calf thymus polyadenosine diphosphate ribose polymerase

The mechanism of poly ADPR synthesis and the transfer of poly ADPR to histone H1 molecule by electrophoretically homogenous calf thymus poly ADPR polymerase containing DNA was examined. 1) An acid insoluble radioactive complex (I) was obtained after incubation of purified enzyme with [3H] NAD. The stability of (I) was examined by SDS-polyacrylamide gel electrophoresis. The complex (I) was stable against acid, SDS, urea, DNase and RNase, but labile against pronase, trypsin, alkali and snake venom phosphodiesterase treatment. The molecular weight of (I) was about 130 000 daltons estimated by SDS-gel electrophoresis. The radioactive products of successive alkali, venom phosphodiesterase and Pronase hydrolysis of (I) were PR-AMP and AMP. The mean chain length of poly ADPR of (I) was 20--30. These results suggest that the complex (I) is poly ADP-ribosylated poly ADPR polymerase. 2) Besides (I), a second radioactive peak (II) was observed when acid insoluble products obtained from an incubation mixture containing purified poly ADPR polymerase, [3H] NAD and purified histone H1 were analyzed on SDS-polyacrylamide gel electrophoresis. The molecular weight of (II) was estimated to be about 23 000 daltons. The complex (II) is eluted like histone H1 on CM-cellulose columns and hydrolyzed by alkali, trypsin and snake venom phosphodiesterase but not by DNase, or RNase. The comples (II) was extracted selectively by 5 per cent perchloric acid or 5 per cent trichloroacetic acid from mixture of (I) and (II). The mean chain length of poly ADPR of complex (II) and 5--20; these results suggest that the complex (II) is poly ADP-ribosylated histone H1. 3) Results 1) and 2) indicate that purified DNA containing, thus DNA independent, poly ADPR polymerase catalyzes two different reactions, the ADPR transfer onto the enzyme itself and onto histone H1 and the elongation of ADPR chains. Dimeric forms of ADP-ribosylated histone H1 was not observed. Free poly ADPR was observed only when very small quantities of enzyme were used for incubation.     

Highly selective affinity labeling of DNA-dependent RNA-polymerase II from human placenta

RNA polymerase II from human placenta was affinity labelled in crude preparation using two-step technique, which includes treatment of the enzyme with an aldehyde-containing reactive analogue of ATP, ADP or AMP in the presence of poly[d(A-T)] followed (after borohydride reduction) by the elongation of the attached label with [alpha-32P]UTP. A polypeptide of the molecular mass ca. 140 kDa proved to be the labelling target. No labelling was observed in the absence of poly[d(A-T)] or the reagent or in the presence of alpha-amanitin. All the results suggest the attachment of the affinity reagents to the second-largest subunit of the human RNA polymerase II, which therefore takes part in the initiation substrate's binding.     

The presence of polyriboadenylic acid sequences in calf lens messenger RNA

The presence of poly(rA) sequences in lens RNA has been demonstrated by the isolation of RNase A and T₁-resistant fragments of approximately 50 nucleotide residues. These poly(rA)-rich sequences, obtained from lenses incubated for six hours in organ culture with [³H]adenosine, are located at the 3′ termini of mRNA as determined by 3′ exoribonuclease digestion. Limited digestion of the [³H]adenosine-labeled mRNA with the enzyme led to the abolition of binding to poly(rU)-filters and a concomitant loss of template activity with avian myeloblastosis virus RNA-dependent DNA polymerase. Furthermore, after incubation of lenses in organ culture with 3′-deoxyadenosine, the isolated polysomal RNA was unable to function as a template in an avian myeloblastosis virus RNA-dependent DNA polymerase-catalyzed reaction system.