Identification and organization of chloroplastic and cytosolic L-myo-inositol 1-phosphate synthase coding gene(s) in Oryza sativa: comparison with the wild halophytic rice, Porteresia coarctata.
The gene coding for rice chloroplastic L-myo-inositol-1-phosphate synthase (MIPS; EC 5.5.1.4) has been identified by matrix-assisted laser desorption time-of-flight mass spectrometry analysis of the purified and immunologically cross-reactive approximately 60 kDa chloroplastic protein following two-dimensional polyacrylamide gel electrophoresis, which exhibited sequence identity with the cytosolic MIPS coded by OsINO1-1 gene. A possible chloroplastic transit peptide sequence was identified upstream of the OsINO1-1 gene upon analysis of rice genome. RT-PCR and confocal microscope studies confirmed transcription, effective translation and its functioning as a chloroplast transit peptide. Bioinformatic analysis mapped the chloroplastic MIPS (OsINO1-1) gene on chromosome 3, and a second MIPS gene (OsINO1-2) on chromosome 10 which lacks conventional chloroplast transit peptide sequence as in OsINO1-1. Two new PcINO1 genes, with characteristic promoter activity and upstream cis-elements were identified and cloned, but whether these proteins can be translocated to the chloroplast or not is yet to be ascertained. Electrophoretic mobility shift assay carried out with nuclear extract of Porteresia coarctata leaves grown under both control and stressed condition shows binding of nuclear proteins with the upstream elements. Nucleotide divergence among the different Oryza and Porteresia INO1 genes were calculated and compared.
Ray
Sudipta
S
Plant Molecular and Cellular Genetics, Bose Institute (Centenary Campus), Kolkata, India.
Patra
Barunava
B
Das-Chatterjee
Aparajita
A
Ganguli
Arnab
A
Majumder
Arun Lahiri
AL
D000595:Amino Acid Sequence; D001483:Base Sequence; D060365:Chloroplast Proteins; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics*; D018388:Codon, Terminator / Q000235:genetics; D003600:Cytosol / Q000201:enzymology*; D030541:Databases, Genetic; D024202:Electrophoretic Mobility Shift Assay; D019143:Evolution, Molecular; D017343:Genes, Plant / Q000235:genetics*; D005966:Glucuronidase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D007296:Myo-Inositol-1-Phosphate Synthase / Q000737:chemistry / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics; D012275:Oryza / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D021382:Protein Sorting Signals / Q000235:genetics; D040901:Proteomics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D060365:Chloroplast Proteins; D018388:Codon, Terminator; D021382:Protein Sorting Signals; C078737:chloroplast transit peptides; D049452:Green Fluorescent Proteins; D005966:Glucuronidase; D007296:Myo-Inositol-1-Phosphate Synthase