Isolation and characterisation of a cDNA clone for a chlorophyll synthesis enzyme from Euglena gracilis. The chloroplast enzyme hydroxymethylbilane synthase (porphobilinogen deaminase) is synthesised with a very long transit peptide in Euglena.
A cDNA expression library was constructed from light-grown Euglena gracilis poly(A)-rich RNA in lambda gt11. Antibodies to Euglena hydroxymethylbilane synthase, the third enzyme in the porphyrin biosynthetic pathway, were used to screen the library and a clone encoding part of the sequence of hydroxymethylbilane synthase was identified. This was used to rescreen the library and a full-length clone was isolated, which encoded not only the entire mature protein (Mr 36,927), but also an N-terminal extension of 139 amino acids. The deduced Mr of the whole polypeptide is 51,744, which corresponds to the size of the protein immunoprecipitated from the translation products of Euglena poly(A)-rich RNA. The mature protein is 60-70% similar to hydroxymethylbilane synthase from human erythrocytes and Escherichia coli. The sequence of the N-terminal extension has similarities to both the transit peptides of chloroplast proteins and those for the endoplasmic reticulum. This is the first report both of a cDNA clone for an enzyme of the chlorophyll biosynthetic pathway and of a putative transit peptide for a nuclear-encoded Euglena protein.
Sharif
A L
AL
Department of Botany, University of Cambridge, England.
Smith
A G
AG
Abell
C
C
D000595:Amino Acid Sequence; D000642:Ammonia-Lyases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002734:Chlorophyll / Q000096:biosynthesis; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005056:Euglena gracilis / Q000201:enzymology / Q000235:genetics*; D015723:Gene Library; D011163:Hydroxymethylbilane Synthase / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D012333:RNA, Messenger; D002734:Chlorophyll; D011061:Poly A; D012313:RNA; D004247:DNA; D011163:Hydroxymethylbilane Synthase; D000642:Ammonia-Lyases