Functional identification of a Delta(8)-sphingolipid desaturase from Borago officinalis

Citation
P. Sperling et al., Functional identification of a Delta(8)-sphingolipid desaturase from Borago officinalis, ARCH BIOCH, 388(2), 2001, pp. 293-298
Citations number
21
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
ISSN journal
0003-9861 → ACNP
Volume
388
Issue
2
Year of publication
2001
Pages
293 - 298
Database
ISI
SICI code
0003-9861(20010415)388:2<293:FIOADD>2.0.ZU;2-I
Abstract
The similarities between Delta (12)- and Delta (15)-fatty acyl desaturase s equences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds. Screening of a bora ge seed cDNA library with an amplified DNA fragment resulted in the isolati on of a full-length cDNA corresponding to a deduced open-reading frame of 4 46 amino acids. The protein showed high similarity to plant Delta (8)-sphin golipid desaturases as well as to the Delta (6)-fatty acyl desaturase from Borago officinalis. The sequence is characterized by the presence of a N-te rminal cytochrome b(5) domain. Expression of this open-reading frame in Sac charomyces cerevisiae resulted in the formation of Delta (8)-trans/cis-phyt osphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives. GLC-MS analysis of the methylated di-0-trimethylsilyl ether derivatives confirmed the presence of Delta (8)-stereoisomers of C-18- and C-20-phytosphingenine. Furthermore, No rthern blotting showed that the gene encoding a stereo-unselective Delta (8 )-sphingolipid desaturase is primarily expressed in young borage leaves. (C ) 2001 Academic Press.