Abstract
A simple strategy has been devised to identify the gene encoding the RNA subunit of RNase P from the zebrafish, Danio rerio. The sequence obtained by amplification of genomic DNA with primers based on sequences common to two other vertebrates was confirmed by reverse transcription and amplification of RNA from a partially purified preparation of the holoenzyme. The 5' and 3' ends were determined by cyclizing the RNA, followed by reverse transcription and sequencing across the ligated RNA junction. The zebrafish sequence is 63% identical to that of Xenopus laevis nuclear RNase P RNA and 69% identical to the human RNase P RNA. A consensus secondary structure was constructed based on these nucleotide identities and on the many compensatory base changes in several regions among these three RNAs. The strategy used to obtain the zebrafish sequence should be useful in deriving analogous gene sequences from diverse classes of eukaryotes.
Highlights
We have identified the full-length, zebrafish RNase P RNA subunit by first isolating a fragment of the corresponding gene (RPR1) from zebrafish genomic DNA by PCR
The genomic DNA fragment is identical in sequence to that obtained from the RNA isolated from partially purified zebrafish RNase P holoenzyme
This confirms that the RPR1 gene that we initially identified is not a pseudogene, but one that is actively transcribed into RNA in order to form the RNase P holoenzyme
Summary
The product of the reaction was run in an 8% polyacrylamide gel under denaturing conditions and aligned with the DNA sequence of the RPR1 clone derived from cyclized RPR1 RNA, in order to identify the nucleotide at the ϩ1 position. Four short regions of identity between the human and X. laevis RNase P RNA genes (see Fig. 1) were identified and, using these sequences, short, nested PCR primers for amplification of zebrafish genomic DNA were designed.
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