The tRNA^Tyr multigene family of Triticum aestivum: genome organization, sequence analyses and maturation of intron‐containing pre‐tRNAs in wheat germ extract

Abstract

Southern analysis of Triticum DNA has revealed that nuclear tRNA Tyr genes are dispersed at a minimum of 16 loci in the genome. We have isolated six independent tRNA Tyr genes from a Triticum aestivum library in addition to three known members of the Triticum tRNA Tyr family. Four of the sequenced tRNA Tyr genes code for Triticum tRNA~ yr and two code for tRNA2 Tyr. Three genes encode tRNAs Ty~ which carry one or two nucleotide substitutions as compared to the conventional genes. The nine Triticum tRNA Ty~ genes possess highly conserved intron sequences ranging in size from 12 to 14 nucleotides. A common secondary intron structure with the 5' and 3' splice site loops separated by five base pairs can be formed by all pretRNAs Tyr which are efficiently spliced in the homologous wheat germ extract.

Characterization of nuclear tRNATyr introns: their evolution from red algae to higher plants

“…Cytoplasmic tRNAs Tyr in higher plants as diverse as Arabidopsis [8], Lupinus [18], Nicotiana [9, 19] and Triticum [7, 20] exhibit identical nucleotide sequences with the exception of one base pair at the basis of the TΨC stem: tRNA Tyr 1 has an A:U pair and tRNA Tyr 2 has a G:C pair at the corresponding position. In order to identify tRNA Tyr genes by PCR analysis in green plants representing Gymnospermae ( Ginkgo ), Pteridophyta ( Rumohra ), Bryophyta ( Polytrichum , Marchantia ) and Charophyta ( Chara ) we employed primers Tyr 1 and Tyr 2, based on 5′ and 3′ distal sequences of tRNA Tyr 1 .…”

“…The reason for the conservation beyond the division is not known. Surprisingly, in almost all other plant species including Triticum [7], Arabidopsis [8] and Nicotiana [9] a uridine is present exclusively at the 5′ end of the intron and a tendency for the occurrence of longer introns ranging in size from 20 to 25 bp can be seen in some higher plants ( Ginkgo and Nicotiana ).…”

“…We have previously isolated numerous nuclear tRNA Tyr genes from genomic libraries of Triticum [7], Arabidopsis [8] and Nicotiana [9]. Here we have characterized additional tRNA Tyr genes from one higher plant ( Pisum ) and eight phylogenetically divergent lower plant species from red algae to gymnosperms employing polymerase chain reaction (PCR).…”

Plant in Vitro Transcription Systems

“…Wheat germ extracts were then shown to accurately process, splice, and modify the pre-tRNA Tyr transcribed in HeLa extracts (35). Human and yeast cell extracts were also shown to transcribe several other plant tRNA genes (2,4,29,36,38,39).…”

A tobacco nuclear extract supporting transcription, processing, splicing and modification of plant intron‐containing tRNA precursors

“…For example, a translational suppression method has made possible the analysis of tRNA transcription in vivo without using promoter fusion vectors (Carneiro et al ., 1993; Choisne et al ., 1997a, b; Ulmasov and Folk, 1995). Furthermore, a cell‐free S23 extract from wheat germs supported accurate processing, modification and splicing in vitro , and using this system many reactions were reported, including the partial purification of an RNase P‐like enzyme (Akama et al ., 2000; Arends and Schön, 1997; Arends et al ., 1996; Beier et al ., 1991; Fuchs et al ., 1992; Junker et al ., 1997; Stange and Beier, 1987; Stange et al ., 1988, 1991, 1992; van Tol et al ., 1987). However, the wheat germ system does not support template‐dependent transcription of tRNA genes (Flynn et al ., 1987; Furter and Hall, 1991).…”