Genomic organization and organ-specific expression of a nuclear gene encoding phage-type RNA polymerase in Nicotiana sylvestris.
We have isolated and sequenced a nuclear gene and cDNA encoding bacteriophage T7-type single subunit RNA polymerase, NsRpoT-A, from Nicotiana sylvestris. NsRpoT-A consists of 19 exons and 18 introns; the first intron is 17 kb, the longest yet identified in a plant gene. Genomic Southern analysis indicated that N. sylvestris contains a small family of NsRpoT genes. Quantitative RT-PCR revealed that steady-state mRNA levels are highest in the leaves and lowest in the cotyledons. Phylogenetic analysis of NsRpoT-A and the RpoT proteins of other plant species suggested that NsRpoT-A is a mitochondrial protein. The TargetP program predicted localization of the NsRpoT-A gene product to the mitochondria. Using a transient expression assay and protoplasts from N. sylvestris mesophyll cells, we clearly demonstrated that the N-terminal sequence of NsRpoT-A targets the protein to the mitochondria. We therefore named this protein NsRpoTm.
Kobayashi
Y
Y
Graduate School of Human Informatics, Nagoya University, 464-8602, Nagoya, Japan.
Dokiya
Y
Y
Sugiura
M
M
Niwa
Y
Y
Sugita
M
M
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology; D001692:Biological Transport; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D049452:Green Fluorescent Proteins; D007438:Introns; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D018744:DNA, Plant; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012321:DNA-Directed RNA Polymerases