Purification and properties of poly(ADP-ribose) synthetase from mouse testicle

Poly(ADP-ribose) synthetase has been purified to apparent homogeneity from mouse testicle by a rapid and simple procedure using column chromatography on DNA-agarose and on Cibacron blue F₃G-A-Sephadex G-150. The purified enzyme absolutely requires DNA for activity, and half-maximal activation occurs at a DNA concentration of 25 μg/ml. The K_m for NAD and V at pH 8.0 and 25 °C are 47 μm and 1400 nmol/min/ mg, respectively. The molecular weight is 116,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicates that the mouse testicle enzyme is very similar to calf thymus enzyme, but there is a difference in the contents of several amino acid residues between the two enzymes. This difference appears to reflect species or tissue specificity of poly(ADP-ribose) synthetase.     

Size of poly (A) +-mRNA in Saccharomyces cerevisiae polysomes.

The size of poly (A) +-mRNA in different classes of yeast polysomes is estimated. The average molecular weight of long-term labelled polysomal poly (A) +-mRNA is about 0,65 x 10(6) daltons. Approximately 60% of the poly (A) +-mRNA polynucleotide chains located at the 5' end, are unprotected by ribosomes and degraded by nucleases upon incubation of cell lysates, to yield a population of poly (A) +-mRNA with an average molecular weight of 0,25 x 10(6) daltons.     

Asymmetrical surface IgG on MOPC-21 plasmacytoma cells contains membrane heavy chain and one secretory heavy chain

Membrane proteins from the B lymphomas WEHI-231 and 2PK3 and from the plasmacytomas MPC-11 and MOPC-21 were radioiodinated in situ by the lactoperoxidase method and were subjected to two-dimensional (nonreduced, reduced) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Few heavily labeled membrane proteins were composed of disulfide-bonded subunits. One such protein (m.w. 200,000 intact and 116,000 reduced) shared some properties with the PC-1 alloantigen, although it was not conclusively identified. A second major disulfide-bonded protein (m.w. 200,000 intact and 95,000 reduced) has been identified previously as the receptor for transferrin. Membrane immunoglobulins of WEHI-231 (IgM) and 2PK3 (IgG2a) had the expected subunit structure, whereas membrane immunoglobulin was not detected on MPC-11. In contrast, surface IgG1 of MOPC-21 appeared to consist almost entirely of hybrid molecules containing one membrane gamma 1 chain and one secretory gamma 1 chain. This hybrid IgG molecule appeared to exist in both monomeric and dimeric forms. It is concluded that i) the synthetic and assembly mechanisms of secretory and membrane IgG1 are shared; ii) there are no special mechanisms to prevent pairing of membrane and secretory gamma 1 chains; iii) the presence of one hydrophobic tail is sufficient for membrane insertion of gamma 1 chains; and iv) the C-terminal extension cysteine residues of membrane gamma 1 chains in hybrid IgG molecules are either unpaired or may allow the formation of hybrid IgG dimers.     

Analysis of MOPC-315 plasmacytoma by elutriation and flow cytometry

Intravenously transplanted murine plasmacytoma MOPC-315 cells were separated from normal spleen cells from a tumour-bearing mouse by elutriation and characterized according to morphology, immunologic properties and clonogenicity. Morphologically, both lymphocytoid and plasmacytoid cells were separable by elutriation. Flow cytometry correlated DNA content and intracytoplasmic IgA content and demonstrated two distinct populations, both in cell cycle, but with markedly different cellular IgA levels. Density gradient separation characterized the lower-density cells with lower IgA content and higher clonogenicity. From these studies a model of cellular differentiation is proposed.     

Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus

Poly(ADP-ribose) glycohydrolase has been purified about 12 300-fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 mumol min -1 mg protein -1. The molecular weight was estimated to be 59 000 by gel filtration through Sephadex G-100 in a non-denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weight, 61 500 and 67 500. The Km value for poly(ADP-ribose) is estimated to be 1.8 microM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP-ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP-ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single-stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double-stranded DNA was not inhibitory.     

Synthesis and coding properties of poly(c 1 A), poly(c 3 A), poly(c 7 A) and poly(h 6 A)

The homopolynucleotides poly(c¹A), poly(c³A), poly(c⁷A) and poly(h⁶A)⁺⁺ were synthesized from their corresponding nucleoside diphosphates using polynucleotide phosphorylase. With the exception of poly(h⁶A), which displayed no hypochromicity, the homopolynucleotides showed melting profiles similar to poly(A). All these polynucleotides, poly(h⁶A), poly(c⁷A), poly(c³A) and poly(c¹A) stimulated the binding of Lys-tRNA to ribosomes; the coding activity of poly(c¹A), however, was very low. Poly(h⁶A) was found to be less specific for Lys-tRNA than poly(A). The data supports the exclusive formation of Watson-Crick type base pairs and contradicts Hoogsteen base pairing in codon-anticodon recognition. Since, however, poly(h⁶A), which can form only one hydrogen bridge per base pair, stimulated the binding of Lys-tRNA comparably to poly(A), the coding activity of the homopolynucleotides tested is discussed in respect to their secondary structure as well as to the pK-values of their 6-amino groups.     

Quantitation and characterisation of poly(A)-containing messenger RNAs from mouse neuroblastoma cells

Comparison of several isolation procedures for neuroblastoma poly(A)-containing mRNAs shows that the highest percentage recovery of undegraded and biologically active messenger RNAs is obtained using proteinase K prior to phenol extraction. The messenger RNAs thus isolated comprise approximately 1.5% of the total ribosomal RNAs and have negligible contamination with 18 and 28 S RNAs. On denaturing polyacrylamide gels they have an average molecular weight of 6.5-10(5) with a range from 2.2-10(5) to 1.53-10(6). The messenger RNAs have an average poly(A) content of 154 nucleotides. They are highly active in wheat germ in vitro protein synthesizing systems, giving as much as 4.3 pmol [35S]methionine incorporation into total protein per mol of mRNA. This is almost as active as a control globin mRNA preparation.     

Complexity of poly(A+) and poly(A-) polysomal RNA in mouse liver and cultured mouse fibroblasts.

RNA excess hybridization experiments were used to measure the complexity of nuclear RNA, poly(A+) mRNA, poly(A-) mRNA, and EDTA-released polysomal RNA sedimenting at less than 80 S in mouse liver and in cultured mouse cells. With both cell types, poly(A-) RNA was found to contain 30-40% of the sequence diversity of total mRNA. In the case of liver this represents 5,700 poly(A-) molecules and 8,600 poly(A+) molecules for a total of approximately 14,300 different mRNAs. Comparison of the complexity of mRNA with that of nuclear RNA revealed that in liver and in cultured cells, mRNA has only 10-20% of the sequence diversity present in nuclear RNA. This latter observation is consistent with existing data on mammalian cells from this and other laboratories.     

Isolation and characterization 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 cryptobiotic gastrulae by ion-exchange chromatography on DEAE-cellulose, DEAE-Sepharose CL-6B and phosphocellulose P11, gel filtration on CL-Sepharose 6B, affinity chromatography on poly(A)-Sepharose 4B and ATP-agarose. The enzyme is fully dependent on exogeneous oligo(riboadenylic acid) and is free of any nuclease or other enzyme activities. In standard assay conditions the enzyme preparation has a specific activity of 5.6 mumol AMP . h-1 . (mg protein)-1. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals the presence of only two proteins with Mr 94 000 and 70 000. The Mr-70 000 protein has been identified as poly(A) polymerase. The enzyme is exclusively activated by Mn2+. Addition of Ca2+, Mg2+, Zn2+, NH4+, K+ or Na+ inhibits the enzymatic reaction. The activity is specific for ATP and competitive inhibition is observed in the presence of other ribonucleoside 5'-triphosphates. AMP incorporation is time-dependent and is increased non-linearly with protein and primer concentration.     

Inhibition of antibody production from the plasmacytoma cell line MOPC-315 by staphylococcal enterotoxin B-induced T-suppressor cells

Our previous results have shown that staphylococcal enterotoxin B (SEB) induces a population(s) of T cells which has the capacity to suppress the antibody response of splenocytes in vitro. In the present report we have attempted to investigate the effect of SEB-primed cells on the secretion of antibody by the plasmacytoma cell line MOPC-315. We have found that the secretion of antibody by MOPC-315 is significantly reduced in as little as 24 hr of coculture with the suppressor cells. The suppressive activity is not antigen- or isotype-specific, since the antibody secretion by both MPC-11 and HOPC1 plasmacytomas are also inhibited by the SEB-primed cells. In addition, we have found that the SEB-primed cell population which inhibits the antibody production by the MOPC-315 cell line expresses the Lyt-1+,2- and Thy-1+ cell surface markers. The apparent relationship between the SEB-primed suppressor cell population and the population which inhibits a conventional antibody response is discussed.     

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.     

A multifunctional RNA recognition motif in poly(A)-specific ribonuclease with cap and poly(A) binding properties

Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3' exoribonuclease that efficiently degrades mRNA poly(A) tails. Here we show that the RNA recognition motif (RRM) of PARN harbors both poly(A) and cap binding properties, suggesting that the RRM plays an important role for the two critical and unique properties that are tightly associated with PARN activity, i.e. recognition and dependence on both the cap structure and poly(A) tail during poly(A) hydrolysis. We show that PARN and its RRM have micromolar affinity to the cap structure by using fluorescence spectroscopy and nanomolar affinity for poly(A) by using filter binding assay. We have identified one tryptophan residue within the RRM that is essential for cap binding but not required for poly(A) binding, suggesting that the cap- and poly(A)-binding sites associated with the RRM are both structurally and functionally separate from each other. RRM is one of the most commonly occurring RNA-binding domains identified so far, suggesting that other RRMs may have both cap and RNA binding properties just as the RRM of PARN.