Molecular characterization and expression study of a histidine auxotrophic mutant (his1-) of Nicotiana plumbaginifolia.
The histidine auxotroph mutant his 1(-) isolated from Nicotiana plumbaginifolia haploid protoplasts was first characterized to be deficient for the enzyme histidinol phosphate aminotransferase that is responsible for one of the last steps of histidine biosynthesis. Expression of the mutated gene at the RNA level was assessed by northern analysis of various tissues. Transcriptional activity was unimpaired by the mutation and, in contrast, a higher level of expression was obtained when compared to the wild-type. The cDNA sequence encoding the mutated gene was isolated by RT-PCR and compared to the wild-type gene. A single point mutation corresponding to the substitution of a G nucleotide by A was identified at position 1212 starting from the translation site. The alignment of the deduced amino acid sequences from the mutated and wild-type gene showed that this mutation resulted in the substitution of an Arg by a His residue at position 381. This Arg residue is a conserved amino acid for histidinol phosphate aminotransferase of many species. These results indicate that the identified mutation results in an altered histidinol phosphate aminotransferase enzyme that is unable to convert the substrate imidazole acetol phosphate to histidinol phosphate and thereby leads to the blockage of histidine biosynthesis. Possible consequences of this blockage on the expression of other amino acid biosynthesis genes were evaluated by analysing the expression of the dhdps gene encoding dihydrodipicolinate synthase, the first key enzyme of the lysine pathway.
El Malki
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Laboratorium voor Plantengenetica, Instituut voor Moleculaire Biologie en Biotechnologie, Vrije Universiteit Brussel, Sint-Genesius Rode, Belgium. mjacobs@vub.ac.be
Jacobs
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D000595:Amino Acid Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005838:Genotype; D006639:Histidine / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009154:Mutation; D010947:Plants, Toxic; D018749:RNA, Plant / 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* / Q000254:growth & development; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D018749:RNA, Plant; D006639:Histidine; D000637:Transaminases; C053430:histidinol-phosphate aminotransferase