An approach towards experimental cDNA sequence determination of predicted genes: an example from Arabidopsis U3-55k homologues.
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.
Urbánek
Pavel
P
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, CZ 166 37 Prague, Czech Republic.
Paces
Jan
J
Paces
Václav
V
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000502:physiology*; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism; D008969:Molecular Sequence Data; D040641:Quantitative Trait Loci / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology; D012335:RNA, Ribosomal / Q000378:metabolism; D020718:Ribonucleoproteins, Small Nucleolar / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods; D017386:Sequence Homology, Amino Acid
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D012335:RNA, Ribosomal; D020718:Ribonucleoproteins, Small Nucleolar; C117377:ribonucleoprotein, U3 small nucleolar; D006655:Histone Deacetylases