Failure to detect "cap" structures in mitochondrial DNA-coded poly(A)-containing RNA from HeLa cells

The structure of the 5'-termini has been investigated in mitochondrial DNA- coded poly(A)-containing RNA from HeLa cells. For this purpose, mitochondrial RNA isolated from cells labeled for 3 hours with [32P]orthophosphate in the presence of 20 µg/ml camptothecin, and selected for poly(A) content by two passages through oligo(dT)-cellulose, was digested either with the nuclease P1 or with a mixture of RNases: the digestion products were then fractionated by two-dimensional electrophoresis. No "cap" structures were detected under conditions where the presence of such structures in one out of five to ten RNA molecules would have been recognized. It is, therefore, likely that "cap" structures are completely absent in HeLa cell mitochondrial poly(A)-containing RNA

Pellicular silicone resins as solid supports for peptide synthesis

Pellicalar si11cone resins were prepared by polymerization of p-bromomethyl phenyl trichlorosilane (I) and 3-(p-chloromethyl phenyl)-1-tridilorosilyl propane (II) on the surface of glass or silica particles. Monctner (I) was synthesized by reaction of p-tolyl magnesium bromide with silicon tetrachloride, follcvzed by radical bromination with N-bromosuccinimide. Monomer (II) was prepared by addition of p-allylbenzyl chloride to trichlorosilane catalyzed by hexachlorcplatinic acid. A tetrapeptide H-Pro-Gly-Phe-Ala-CH was synthesized on the silicone support fran monomer (I) by standard Merrifield procedure. No failure sequences were detected by amino acid analysis and thin layer chranatography. The cleavage of the peptide by HBr-TFA did not proceed to coupletion. The dodecapeptide H-(Phe-Ala)[lowered 6]OH was syntliesized on the support from monomer (II) by the same procedure. Cleavage by HBr-TFA was in this case practically complete. The peptide was analyzed for the presence of failure sequences by partial hydrolysis with 12 N HCl, followed by GC-MS analysis. Finally, pentapeptide H-GlN-GlN-Gly-Gly-Tyr-NH[lowered 2] was prepared on the support fran monaner (II) by column procedure. The peptide was cleaved fran the resin as a methyl ester, by action of triethylamine/methanol. The protected peptide was purified by gel permeation chranatography and amidated with NH[lowered 3] in DMF-MeOH mixture. The Boc-group was cleaved with INHCl/AcOH. The benzyl groups protecting the side chain of tyrosine were cleaved by catalytic hydrogenation over Pd/charcoal.Chemistry, Department o

Separation of oligonucleotides by thin-layer electrophoresis

A method for fingerprinting of ^(32)P-labeled oligonucleotides is described. It involves separation by electrophoresis on cellulose thin layers in the first dimension, followed by chromatography on DEAE cellulose in the second dimension. The main advantages of the method are improved separation and simplicity

Separation of oligonucleotides by thin-layer electrophoresis

A method for fingerprinting of ^(32)P-labeled oligonucleotides is described. It involves separation by electrophoresis on cellulose thin layers in the first dimension, followed by chromatography on DEAE cellulose in the second dimension. The main advantages of the method are improved separation and simplicity

Nucleotide sequences of the 5′ termini of φX174 mRNAs synthesized in vitro

When using φX174 RFI DNA as a template, in vitro, E. coli RNA polymerase synthesizes four major purine triphosphate-containing 5′ end sequences. RNase A digests of α(32)P labeled RNA were further digested with spleen exonuclease to remove the bulk of the oligonucleotides with 5′ hydroxyls and then chromatographed on DEAE cellulose to resolve the remaining 5′ terminal oligonucleotides. By application of standard separation and sequence techniques, the major 5′ end sequences were shown to be: pppApUp(Cp), pppApApApUp(Cp), pppApApApApUp(Cp), and pppGpApUp(Gp)

New method for isolation and sequence determination of 5'-terminal regions of bacteriophage φX174 in vitro mRNAs

We have determined the nucleotide sequences of the 5’-terminal oligonucleotides, produced by RNase T_1 digestion of bacteriophage φX174 mRNAs synthesized in vitro. The major sequences are: pppCpGp(Ap), pppApUpCpGp(Cp), pppAp(Ap)_2UpCp(Up)_2Gp(Gp), and pppAp(Ap)_3UpCp(Up)_2Gp(Gp). The sequences of several minor 5’-terminal oligonucleotides were also determined. For this research we have devised a simple isolation procedure, for the 5’-terminal oligonucleotides, based upon hydroxylapatite chromatography and two-dimensional thin-layer separation. This method allows for the rapid and quantitative recovery of all oligonucleotides containing 5’-triphosphate end groups and should be generally useful for sequence on 5’ termini of mRNAs