Complementation cloning and sequence analysis of the Chlamydomonas reinhardtii hemL gene encoding glutamate-1-semialdehyde aminotransferase

Glutamate-1-semialdehyde amino-transferase (GSAT) catalyzes formation of the tetrapyrrole precursor, [delta]-aminolevulinic acid. GSAT is encoded by the hemL gene. A Chlamydomonas reinhardtii hemL cDNA was selected from a vegetative stage expression library by complementation of Escherichia coli hem...

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Bibliographic Details
Main Authors: Matters, G.L., Beale, S.I.
Format: Conference Proceeding
Language:English
Subjects:
ALGAE
AMINO ACIDS
AMINOLEVULINIC ACID
AMINOTRANSFERASES
BACTERIA
BARLEY
BASIC BIOLOGICAL SCIENCES
CARBOXYLIC ACIDS
CEREALS
CHLAMYDOMONAS
CHLOROPHYCOTA
CLONING
DNA HYBRIDIZATION
DNA SEQUENCING
DNA-CLONING
ENZYMES
ESCHERICHIA COLI
GENES
GRAMINEAE
HYBRIDIZATION
LILIOPSIDA
MAGNOLIOPHYTA
MICROORGANISMS
NITROGEN TRANSFERASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
PLANTS
PROTEINS
STRUCTURAL CHEMICAL ANALYSIS
TOBACCO
TRANSFERASES
UNICELLULAR ALGAE 550400 > Genetics
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Summary:Glutamate-1-semialdehyde amino-transferase (GSAT) catalyzes formation of the tetrapyrrole precursor, [delta]-aminolevulinic acid. GSAT is encoded by the hemL gene. A Chlamydomonas reinhardtii hemL cDNA was selected from a vegetative stage expression library by complementation of Escherichia coli hemL mutant GE 1377. In vitro GSAT activity was ten-fold higher in an extract of the complemented hemL cells than in an extract of uncomplemented mutant cells. The complementing cDNA is 2010 bp long and includes 591 bp of 3' noncoding DNA and an estimated 27 bp of 5' noncoding DNA. The coding region includes the sequence for a putative 30-amino acid chloroplast transit peptide and a 433-amino acid mature protein. The mature protein deduced from the Chlamydomonas cDNA sequence has a molecular weight of 45,880, compared to the value of 43,000 reported for purified Chlamydomonas GSAT (d. Jahn et al., 1991, J. Biol. Chem. 266:161-167). The deduced peptide is 74% identical to Synechococcus GSAT, 70% identical to barley GSAT and 66% identical to tobacco GSAT. The putative pyridoxal binding region has the sequence TTMGKVIGG, which differs somewhat from those reported for other aminotransferases. The deduced putative chloroplast transit peptide has recognizable similarity to barley GSAT transit peptide. Southern analysis of genomic DNA from Chlamydomonas strain CC124, using the cDNA as a probe, indicates that GSAT is probably encoded by a single gene.
ISSN:0032-0889
1532-2548