An approach towards experimental cDNA sequence determination of predicted genes: an example from Arabidopsis U3-55k homologues

Gene. 2005 Sep 26:358:67-72. doi: 10.1016/j.gene.2005.05.022.

Abstract

Experimental cDNA sequence determinations lag behind in silico gene structure predictions in some recently sequenced genomes. This may be due in part to low transcript abundance and/or the severely spatio-temporarily restricted expression pattern of some genes. Here we characterize the predicted repressed gene of Arabidopsis thaliana (At4g21130) that encodes a homologue of the Arabidopsis U3-55K-like protein (At4g05410) and of the U3-55K (RNU3IP2, Rrp9p) proteins from other eukaryotes. In man and yeast, U3-55K is involved in the processing of the pre-ribosomal RNA. Here we show that treatment with inhibitors of histone deacetylases (trichostatin A, sodium butyrate) or DNA methyltransferases (5-aza-2'-deoxycytidine) induces a low but distinct level of mRNA from the repressed Arabidopsis At4g21130 locus, which can be detected by RT-PCR amplification. Direct sequencing of PCR products reveals the open reading frame that differs, in part, from the hypothetical one and encodes a seven-WD-repeat protein highly conserved when compared to U3-55K proteins from various eukaryotic species. This suggests the conservation of its function. The described approach may help to determine the nucleotide sequences of transcripts from predicted genes with a low level of expression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / genetics*
  • Arabidopsis / metabolism
  • Arabidopsis Proteins / genetics*
  • Arabidopsis Proteins / metabolism
  • Cloning, Molecular
  • DNA, Complementary / genetics*
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation, Plant / drug effects
  • Gene Expression Regulation, Plant / physiology*
  • Histone Deacetylase Inhibitors
  • Histone Deacetylases / metabolism
  • Molecular Sequence Data
  • Quantitative Trait Loci / genetics
  • RNA Processing, Post-Transcriptional / physiology
  • RNA, Ribosomal / metabolism
  • Ribonucleoproteins, Small Nucleolar / genetics*
  • Ribonucleoproteins, Small Nucleolar / metabolism
  • Sequence Analysis, DNA / methods
  • Sequence Analysis, Protein / methods
  • Sequence Homology, Amino Acid

Substances

  • Arabidopsis Proteins
  • DNA, Complementary
  • Enzyme Inhibitors
  • Histone Deacetylase Inhibitors
  • RNA, Ribosomal
  • Ribonucleoproteins, Small Nucleolar
  • ribonucleoprotein, U3 small nucleolar
  • Histone Deacetylases