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The characteristics of the enzyme of porcine retrovirus from a cell line derived from swine malignant lymphomas fit with the template specificity and divalent cation requirement for reverse transcriptase. It is further found that the virus particle contains a high molecular mass 70 S RNA as viral genome.
Two fragments (21-mers) containing consensus sequences for the self-cleavage domain in transcripts of satellite DNA of the newt were chemically synthesized and found to be cleaved in the presence of Mg2+. The cleaved product contained the 3'-terminal 2',3'-cyclic phosphate. Twenty-five combinations of partially double-stranded 21-mer RNA which contained different bases within the consensus sequences and at the cleavage sites were tested for self-cleavage. It seemed that guanosine 3'-phosphate was not susceptible to transesterification at the cleavage site.
We have examined the antithrombin effects of various phosphate-containing polyanions (including linear polyphosphates, polynucleotides and the phosphoserine glycoprotein, phosvitin) on the glycosaminoglycan-binding plasma proteinase inhibitors, antithrombin III (ATIII) and heparin cofactor II (HCII). These phosphate-containing polyanions accelerate the HCII-thrombin reaction, as much as 1600-fold in the case of phosvitin. The HCII-thrombin reaction with both phosvitin and polynucleotides appears to follow the ternary complex mechanism. The HCII-thrombin complex is rapidly formed in the presence of these phosphate polyanions (each at 10 micrograms/ml) when 125I-labeled thrombin is incubated with human plasma (ex vivo). None of these phosphate polyanions accelerate the ATIII-thrombin reaction. Our results suggest that the antithrombotic effect of these phosphate-containing polyanions is mediated by HCII activation and not by ATIII.
A 37-mer hammerhead ribozyme has been designed to efficiently cleave the 1.4 kb mRNA of the urokinase plasminogen activator receptor (uPAR). Under in vitro conditions, the chemically synthesized ribozyme cleaved uPAR mRNA and inhibited its translation in a concentration-dependent fashion. The ribozymes were 5'-[35S]thiophosphorylated and used as a model to analyze conditions for RNA delivery in a cultured human osteosarcoma cell system. Ribozymes degraded immediately in cell-conditioned medium but ribozymes complexed with lipofectin were protected from RNases for up to 22 h. Lipofectin rapidly transported ribozyme into the cell, where it accumulated almost exclusively in the cytoplasm. Thus, lipofectin dramatically enhances stability and cytoplasmic delivery of ribozymes, potentially enabling targeting of mRNA in vivo.
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The binding of [14C] spermine to polynucleotides has been studied by equilibrium dialysis and the data analysed by Scatchard plots. The binding of spermine to poly(A) shows a binding site for 1 spermine/140 nucleotides when measured in 0.2M NaCl at 5 degrees C. Poly(C) also has a similar sites; on the other hand poly(U) and poly(G) each have a binding site for 1 spermine/12 nucleotides. The addition of complementary di- or trinucleotides to either poly(A) or poly(U) affects their ability to bind spermine, in particular the high affinity site on poly(A) is no longer detectable. The effect of spermine, spermidine and putrescine on the binding of polynucleotides to complementary di- and trinucleotides was also studied. Spermine markedly increased the binding of both ApA and of ApApA to poly(U) whereas spermidine and putrescine had very little effect. In contrast spermine had little effect on the binding of either UpU or UpUpU to poly(A). These results suggest that spermine binding to oligo- and polynucleotides is dependent on the particular nucleotide combination involved and that spermine may therefore be able to act selectively within cells.
A direct competitive binding radioimmunoassay for DNA has been developed, using 125I-iododeoxyuridine-labelled DNA as the antigen and the serum from a patient with systemic lupus erythematosus. The assay is sensitive in the range of 25 to 1000 ng/ml of DNA. The sensitivity is determined by the affinity of the antibody: this SLE serum contains a component with an association constant of 9.6 X 10(-5) l/mol active at high dilution (1/10,000). Any biological material, such as serum, synovial fluid or tissue extracts can be tested directly. No interference has been found by DNAse in normal serum, or inhibition by mononucleotides or RNA. Native or denatured DNA from different sources (Escherichia coli, salmon sperm, calf thymus and human placenta) either purified or not, competes equally well for the antibody in this system.
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Poly(C, U) random copolymer templates direct the oligomerization of 2-MeImpG and 2-MeImpA, resulting in the production of a variety of oligo/(G,A)s. The efficiency of monomer incorporation into newly synthesized oligomers is greater for 2-MeImpG than for 2-MeImpA, and decreases for both monomers as the uracil content of the template increases. The relatively poor incorporation of adenine is partly due to an intrinsically less efficient incorporation reaction, and partly due to the masking of uracil sites by G X U non-complementary pairing. The efficiency of adenine incorporation can be improved by decreasing the concentration of 2-MeImpG and increasing the concentration of 2-MeImpA in the reaction mixture. The oligomeric product distribution can be characterized in detail using high-pressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition, and phospho-diester-linkage isomerism. The 3'----5' regiospecificity of monomer addition to template-bound oligomers is lower for 2-MeImpA than for 2-MeImpG. The presence of an adenine residue at the 2'(3') terminus of the acceptor strand lowers the regiospecificity of 2-MeImpA addition even further.