Publisher Summary This chapter describes a general method, which has made possible the sequence analysis of tRNAs from mitochondria, chloroplasts, and a wide variety of prokaryotic and eukaryotic organisms using in some cases as little as 20μg of purified tRNA. The development of in vitro 32 P-labeling techniques has now permitted sequence analysis of nonradioactive tRNAs utilizing adaptations of methods developed by Sanger and co-workers for analysis of uniformly 32 P-labeled RNAs. oligonucleotides with a free 5'-hydroxyl end which are present in digests of nonradioactive nucleic acids can be labeled to high specific activity using T4 polynucleotide kinase and [γ- 32 P]ATP. This approach is used for labeling with 32 P the 5' ends of both mononucleotides present in complete tRNA hydrolysates and oligonucleotides present in T1 or pancreatic RNase digests of tRNA. Using oligonucleotides present in tRNAs of known sequence as standards, the mobility shifts of many commonly occurring modified nucleotides are characterized, which has proved useful in recognizing these modified nucleotides within unknown sequences. The basic steps in the sequence analysis of a nonradioactive tRNA are summarized in the chapter.

Use of in vitro 32P labeling in the sequence analysis of nonradioactive tRNAs.

Sequence analysis of 5'-[32P] labeled tRNA and eukaryotic mRNA using an adaptation of a method recently described by Donis-Keller, Maxam and Gilbert for mapping guanines, adenines and pyrimidines from the 5'-end of an RNA is described. In addition, a technique utilizing two-dimensional polyacrylamide gel electrophoresis for identification of pyrimidines within a sequence is described. 5'-[32P] Labeled rabbit beta-globin mRNA and N. crassa mitochondrial initiator tRNA were partially digested with T1- RNase for cleavage at G residues, with U2-RNase for cleavage at A residues, with an extracellular RNase from B. cereus for cleavage at pyrimidine residues and with T2-RNase or with alkali for cleavage at all four residues. The 5'-[32P] labeled partial digestion products were separated according to their size, by electrophoresis in adjacent lanes of a polyacrylamide slab gel and the location of G's, A's and of pyrimidines extending 60-80 nucleotides from the 5'-end of the RNA determined. Two-dimensional polyacrylamide gel electrophoresis was used to separate the 5'-[32P] labeled fragments present in partial alkali digests of a 5'-[32P] labeled mRNA. The mobility shifts corresponding to the difference of a C residue were distinct from those corresponding to a U residue and this formed the basis of a method for distinguishing between the pyrimidines.

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Sequence analysis of 5′[32P labeled mRNA and tRNA using polyacrylamide gel electrophoresis

Fractionation and identification of spinach chloroplast transfer RNAs and mapping of their genes on the restriction map of chloroplast DNA.

Euglena gracilis chloroplast ribosomal RNA transcription units. II. Nucleotide sequence homology between the 16 S--23 S ribosomal RNA spacer and the 16 S ribosomal RNA leader regions.

W E Barnett

Papers

The first nucleotide sequence of an organelle transfer RNA: Chloroplastic tRNAPhe