scholarly journals Analysis of Small RNAs by Northern Hybridization

2018 ◽  
Vol 2018 (8) ◽  
pp. pdb.prot097493 ◽  
Author(s):  
Chengjian Li ◽  
Phillip D. Zamore
Keyword(s):  
Small Rnas ◽  
Northern Hybridization

Organization and expression of algal ( Chlamydomonas reinhardtii ) mitochondrial DNA

1988 ◽  
Vol 319 (1193) ◽  
pp. 135-147 ◽  
Keyword(s):  
Mitochondrial Dna ◽  
Codon Usage ◽  
Chlamydomonas Reinhardtii ◽  
Small Rnas ◽  
Rrna Genes ◽  
Gene Arrangement ◽  
Transcriptional Unit ◽  
Nucleotide Sequence Analysis ◽  
Northern Hybridization ◽  
Protein Coding

The mitochondrial genome of Chlamydomonas reinhardtii , a unicellular green alga, is a linear 15.8 kilobase pair (kbp) molecule. In gene arrangement and mode of expression, as well as in size, it differs radically from the large (200-2400 kbp) mitochondrial genomes of higher plants. Heterologous hybridization experiments and nucleotide sequence analysis have revealed that C. reinhardtii mitochondrial DNA (mtDNA) is a compactly organized genome specifying at least eight proteins, a minimum of three transfer RNAs, and large subunit (LS) and small subunit (SS) ribosomal RNAs. Both strands of the mtDNA encode genetic information, with genes organized into perhaps a single transcriptional unit on each strand. Stable transcripts have been identified by Northern hybridization analysis, and transcript termini have been mapped by primer extension and SI nuclease protection experiments. The results suggest that mature RNAs, which virtually saturate the genome, are generated by precise endonucleolytic cleavage of long precursors, with specific motifs (both primary sequence and secondary structure) implicated as processing signals. Codon usage in C. reinhardtii mitochondria is highly biased, with eight codons entirely absent from all protein-coding genes; however, even though codon usage is restricted, it appears that C. reinhardtii mtDNA cannot encode the minimum number of tRNAs needed to support mitochondrial protein synthesis. The most striking feature of C.reinhardtii mtDNA is the division of SS and LS rRNA genes into a number of separate subgenic coding segments (‘modules’) that are interspersed with one another and with protein-coding and tRNA genes. We have identified abundant small RNAs, transcribed from these modules, that approximate to the latter in size. This indicates that splicing of rRNA ‘pieces’ does not occur in this system. Rather, the mature rRNAs apparently exist and function as non-covalent complexes of small RNAs (four in SS rRNA, at least eight in LS rRNA), held together by intermolecular base pairing. These complexes contain all the conserved elements of the minimal secondary structures that define the functional core of conventional LS and SS rRNAs.

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