Molecular cloning and expression of a cDNA sequence encoding histidinol phosphate aminotransferase from Nicotiana tabacum.
A Nicotiana tabacum cDNA sequence encoding histidinol phosphate aminotransferase (HPA) was isolated by functional complementation of an Escherichia coli histidine auxotroph (UTH780). The enzymatic assay has confirmed that the isolated cDNA encodes a functional HPA protein. Amino acid sequence alignment of the HPA protein from N. tabacum, Saccharomyces cerevisiae and E. coli revealed that, despite the low degree of identity, some residues were found to be highly conserved. The predicted protein contains a transit peptide sequence at the amino-terminal end, suggesting a chloroplastic localization of the HPA enzyme. Western blot analysis demonstrated that the deduced HPA protein and the mature HPA protein have an apparent molecular mass of about 45 kDa and 40 kDa respectively. Gene copy number estimation by Southern analysis indicates the presence of at least two genes per haploid genome coding for this protein in Nicotiana sp. From northern analysis results, the gene seems to be highly expressed in green tissues and the detected transcript showed a single band of expected molecular size.
El Malki
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F
Laboratory for Plant Genetics, Vrije Universiteit Brussel, Sint-Genesius Rode, Belgium.
Frankard
V
V
Jacobs
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M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D017343:Genes, Plant; D005816:Genetic Complementation Test; D006639:Histidine / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*; D000637:Transaminases / Q000096:biosynthesis / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D018749:RNA, Plant; D011994:Recombinant Proteins; D006639:Histidine; D000637:Transaminases; C053430:histidinol-phosphate aminotransferase