Inhibition of In Vitro Transcription by Adenosine Antibodies

Antibodies against adenosine markedly inhibited in vitro transcription in isolated BHK 21 nuclei in a dose-dependent manner. The inhibition was specific as it could be completely reversed by the addition of homologous hapten. Addition of RNA at low concentration reversed the inhibition, whereas excess DNA did not have any effect. Adenosine antibodies also inhibited in vitro transcription with calf thymus DNA and E. coli RNA polymerase. Antibodies that react with DNA but not with RNA such as anti-dpA, anti-dpC and anti-DNA failed to inhibit in vitro transcription in isolated nuclei as well as with calf thymus DNA and E. coli RNA polymerase. The results strongly indicate that the binding of adenosine antibodies to RNA is responsible for the inhibition of transcription

Inhibition of in Vitro Amino Acylation and Translation by Adenosine Antibodies

Adenosine antibodies markedly inhibited in vitro amino acylation of tRNA in a dose-dependent manner. The inhibition was specific as it was reversed by the homologous hapten. Addition of excess tRNA reversed the inhibition indicating that binding of antibodies to tRNA is responsible for inhibition. Adenosine antibodies also inhibited in vitro translation of endogenous mRNAs in rabbit reticulocyte lysate in a dose-dependent manner. The homologous hapten reversed the inhibition showing thereby the immunospecificity of inhibition

Antibodies raised against denatured DNA bind to double-stranded DNA

Antibodies raised against denatured DNA complexed with methylated bovine serum albumin have been reported to react with ssDNA but not with dsDNA. Using a highly sensitive avidin-biotin microELISA, we report that such antibodies also bind to dsDNA. Antibodies which reacted with ssDNA and dsDNA were found to be IgG type. The antibodies did not react with tRNA and rRNA. The binding of antibodies to dsDNA was partially inhibited dy individual deoxyribonucleotides. ssDNA as well as dsDNA inhibited the binding of antibodies to dsDNA. The binding of these antibodies to supercoiled and relaxed forms of pBR322 DNA was demonstrated by gel retardation assay. The cross-reaction with ssDNA was observed even after affinity purification on native DNA-cellulose. The antibodies were also shown to bind to poly(dA-dT)·poly(dA-dT

Binding of deoxyadenylate and deoxycytidylate antibodies to double-stranded DNA

Antibodies raised against deoxyadenylate and deoxycytidylate were found to react with double stranded DNA as assessed by highly sensitive avidin-biotin microELISA. The binding was specific as it was completely inhibited by the homologous hapten. The antibodies did not react with tRNA and rRNA. These antibodies were also shown to react with supercoiled and relaxed forms of pBR322 DNA as demonstrated by gel retardation assay. ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; CT DNA, calf thymus DNA; AB microELISA, avidin-biotin microELISA; dpA, deoxyadenylate; dpC, deoxycytidylate; avidin-HRP, avidin-horseradish peroxidas

Base-Specific Binding of Adenosine Antibodies to Double-Stranded DNA

Antibodies raised against adenosine have been reported to react with single-stranded DNA but not with double-stranded DNA. Using a highly sensitive avidin-biotin microELISA we report that these antibodies also react with double-stranded DNA. The binding was specific as it was completely inhibited by the homologous hapten. The results indicate that the antibody populations binding to ssDNA and dsDNA are not cross-reactive. The antibodies were shown to react with the topoisomers of plasmid DNA as assessed by gel retardation assay. The antibodies showed differential binding to restriction fragments of DNA indicating that some of the A residues in dsDNA are accessible to the antibodies